Background We have demonstrated that moderate alcohol consumption (15 g/d, 30 g/d) for 8 weeks resulted in significantly increased levels of serum estrone sulfate and DHEAS in 51 postmenopausal women in a randomized, placebo-controlled trial. We now report on the relationships between serum estrone sulfate and dehydroepiandrosterone sulfate (DHEAS) levels after 4 weeks of moderate alcohol supplementation, and compare the results to the 8 weeks data to elucidate time-to-effect differences.
Background
We have demonstrated that moderate alcohol consumption (15 g/d, 30 g/d) for 8 weeks resulted in significantly increased levels of serum estrone sulfate and DHEAS in 51 postmenopausal women in a randomized, placebo-controlled trial. We now report on the relationships between serum estrone sulfate and dehydroepiandrosterone sulfate (DHEAS) levels after 4 weeks of moderate alcohol supplementation, and compare the results to the 8 weeks data to elucidate time-to-effect differences.Methods Postmenopausal women (n = 51) consumed 0 (placebo), 15 (1 drink), and 30 (2 drinks) g alcohol (ethanol)/ day for 8 weeks as part of a controlled diet in a randomized crossover design. Blood samples were drawn at baseline, at 4 weeks and at 8 weeks. Changes in estrone sulfate and DHEAS levels from placebo to 15 g and 30 g alcohol per day were estimated using linear mixed models.
Methods
Postmenopausal women (n = 51) consumed 0 (placebo), 15 (1 drink), and 30 (2 drinks) g alcohol (ethanol)/ day for 8 weeks as part of a controlled diet in a randomized crossover design. Blood samples were drawn at baseline, at 4 weeks and at 8 weeks. Changes in estrone sulfate and DHEAS levels from placebo to 15 g and 30 g alcohol per day were estimated using linear mixed models.Results and Discussion At week 4, compared to the placebo, estrone sulfate increased an average 6.9% (P = 0.24) when the women consumed 15 g of alcohol per day, and 22.2% (P = 0.0006) when they consumed 30 g alcohol per day. DHEAS concentrations also increased significantly by an average of 8.0% (P < 0.0001) on 15 g of alcohol per day and 9.2% (P < 0.0001) when 30 g alcohol was consumed per day. Trend tests across doses for both estrone sulfate (P = 0.0006) and DHEAS (P < 0.0001) were significant. We found no significant differences between the absolute levels of serum estrone sulfate at week 4 versus week 8 (P = 0.32) across all doses. However, absolute DHEAS levels increased from week 4 to week 8 (P < 0.0001) at all three dose levels.
Results and Discussion
At week 4, compared to the placebo, estrone sulfate increased an average 6.9% (P = 0.24) when the women consumed 15 g of alcohol per day, and 22.2% (P = 0.0006) when they consumed 30 g alcohol per day. DHEAS concentrations also increased significantly by an average of 8.0% (P < 0.0001) on 15 g of alcohol per day and 9.2% (P < 0.0001) when 30 g alcohol was consumed per day. Trend tests across doses for both estrone sulfate (P = 0.0006) and DHEAS (P < 0.0001) were significant. We found no significant differences between the absolute levels of serum estrone sulfate at week 4 versus week 8 (P = 0.32) across all doses. However, absolute DHEAS levels increased from week 4 to week 8 (P < 0.0001) at all three dose levels.Conclusions These data indicate that the hormonal effects due to moderate alcohol consumption are seen early, within 4 weeks of initiation of ingestion.
Conclusions
These data indicate that the hormonal effects due to moderate alcohol consumption are seen early, within 4 weeks of initiation of ingestion.

Background Epidemiological evidence consistently shows a positive association between alcohol, even low to moderate intake, and breast cancer risk [ 1 ]. However, during the past two decades, it has become evident that moderate drinking is associated with longer life [ 2 ], reduced rates of heart disease [ 3 ] and stroke [ 4 ]. What does this mean for women when the epidemiologic data show an exposure is associated with both benefits and harms? Recommendations regarding the use or avoidance of moderate alcohol, must take into consideration both its potential benefit on cardiovascular disease, as well as its potential risk for breast cancer. To understand the biologic parameters potentially influenced by alcohol, there is a need for well-controlled mechanistic studies of dose and duration of use on markers of risk in the causal pathway of breast cancer. A reanalysis of nine prospective studies shows that levels of endogenous sex hormones are strongly associated with breast cancer risk in postmenopausal women [ 5 ]. Clear evidence that moderate alcohol consumption increases levels of hormones associated with increased risk for breast cancer would provide support for a causal relationship. In a controlled study of acute alcohol ingestion, serum estrone levels were significantly elevated in postmenopausal women on hormone replacement therapy (HRT), but did not affect serum estrone levels in women not using HRT [ 6 ]. To better understand the effects of moderate long-term alcohol ingestion on sex hormones, we conducted a controlled feeding study in postmenopausal women not using HRT. As previously reported, there were significant elevations in both serum estrogen sulfate and dehydroepiandrosterone sulfate (DHEAS) after 8 weeks of supplementation [ 7 ]. Here, we evaluate the relationships between serum estrogen sulfate and DHEAS after 4 weeks of supplementation with moderate alcohol, and compare the results to the 8 week data to elucidate time-to-effect differences.
Background
Epidemiological evidence consistently shows a positive association between alcohol, even low to moderate intake, and breast cancer risk [ 1 ]. However, during the past two decades, it has become evident that moderate drinking is associated with longer life [ 2 ], reduced rates of heart disease [ 3 ] and stroke [ 4 ]. What does this mean for women when the epidemiologic data show an exposure is associated with both benefits and harms? Recommendations regarding the use or avoidance of moderate alcohol, must take into consideration both its potential benefit on cardiovascular disease, as well as its potential risk for breast cancer. To understand the biologic parameters potentially influenced by alcohol, there is a need for well-controlled mechanistic studies of dose and duration of use on markers of risk in the causal pathway of breast cancer.A reanalysis of nine prospective studies shows that levels of endogenous sex hormones are strongly associated with breast cancer risk in postmenopausal women [ 5 ]. Clear evidence that moderate alcohol consumption increases levels of hormones associated with increased risk for breast cancer would provide support for a causal relationship. In a controlled study of acute alcohol ingestion, serum estrone levels were significantly elevated in postmenopausal women on hormone replacement therapy (HRT), but did not affect serum estrone levels in women not using HRT [ 6 ]. To better understand the effects of moderate long-term alcohol ingestion on sex hormones, we conducted a controlled feeding study in postmenopausal women not using HRT. As previously reported, there were significant elevations in both serum estrogen sulfate and dehydroepiandrosterone sulfate (DHEAS) after 8 weeks of supplementation [ 7 ]. Here, we evaluate the relationships between serum estrogen sulfate and DHEAS after 4 weeks of supplementation with moderate alcohol, and compare the results to the 8 week data to elucidate time-to-effect differences.Methods Details of the Women's Alcohol Study (WAS) was previously published [ 7 ]. Briefly, a total of 51 postmenopausal women not on HRT completed the study and are included in the analysis. The WAS utilized a crossover design. Each participant rotated through three 8-weeks controlled dietary periods during which she consumed a beverage daily that contained no alcohol (placebo), 15 g alcohol, or 30 g alcohol in random order. Each of the three dietary periods was preceded by a 2- to 5-week washout period during which the women consumed no alcohol. Alcohol was supplied as 95% ethanol (Everclear™ Pharmaco Products, Inc., Brookfield, CN) in orange juice (12 ounces). All meals were prepared at the Beltsville Human Nutrition Research Center and the participants ate breakfast and supper at the Center and had carryout lunches on weekdays. On weekends, food and beverages were packaged for consumption at home. The calorie level for each subject was adjusted to maintain constant body weight. Blood for hormone analyses was collected after an overnight fast at weeks 4 and 8. Serum was separated, and aliquots were frozen at -70°C. In the samples taken after 4 weeks of alcohol supplementation, we only measured serum estrone sulfate and DHEAS, as they were the only two hormones that were significantly elevated at 8 weeks [ 7 ]. Hormone concentrations were transformed using natural log. Changes in hormone concentrations from placebo to 15 g and 30 g of alcohol per day were estimated at 4 and 8 weeks using linear mixed models including a random intercept and alcohol levels as fixed effects treated as two indicator variables. Separate models used alcohol as a continuous variable to test for trend. Regressions to evaluate the effect of several baseline covariates on the precision of the parameter estimates for alcohol consumption included age, BMI, and years since menopause modeled as continuous fixed effects; assignment order, dietary period, hysterectomy, and race were modeled as indicator variables. Effect modification by race, assignment order, dietary period, age, BMI, and years since menopause were assessed by likelihood ratio tests of improvement in the model fit after addition of cross-product terms to models that included main effects for alcohol and the characteristic being evaluated. All tests of statistical significance were two-sided. Statistical analyses were performed using S-PLUS (S-PLUS version 6.1 for Windows. Seattle (WA): Insightful Corporation; 2002.)
Methods
Details of the Women's Alcohol Study (WAS) was previously published [ 7 ]. Briefly, a total of 51 postmenopausal women not on HRT completed the study and are included in the analysis. The WAS utilized a crossover design. Each participant rotated through three 8-weeks controlled dietary periods during which she consumed a beverage daily that contained no alcohol (placebo), 15 g alcohol, or 30 g alcohol in random order. Each of the three dietary periods was preceded by a 2- to 5-week washout period during which the women consumed no alcohol. Alcohol was supplied as 95% ethanol (Everclear™ Pharmaco Products, Inc., Brookfield, CN) in orange juice (12 ounces). All meals were prepared at the Beltsville Human Nutrition Research Center and the participants ate breakfast and supper at the Center and had carryout lunches on weekdays. On weekends, food and beverages were packaged for consumption at home. The calorie level for each subject was adjusted to maintain constant body weight.Blood for hormone analyses was collected after an overnight fast at weeks 4 and 8. Serum was separated, and aliquots were frozen at -70°C. In the samples taken after 4 weeks of alcohol supplementation, we only measured serum estrone sulfate and DHEAS, as they were the only two hormones that were significantly elevated at 8 weeks [ 7 ].Hormone concentrations were transformed using natural log. Changes in hormone concentrations from placebo to 15 g and 30 g of alcohol per day were estimated at 4 and 8 weeks using linear mixed models including a random intercept and alcohol levels as fixed effects treated as two indicator variables. Separate models used alcohol as a continuous variable to test for trend. Regressions to evaluate the effect of several baseline covariates on the precision of the parameter estimates for alcohol consumption included age, BMI, and years since menopause modeled as continuous fixed effects; assignment order, dietary period, hysterectomy, and race were modeled as indicator variables. Effect modification by race, assignment order, dietary period, age, BMI, and years since menopause were assessed by likelihood ratio tests of improvement in the model fit after addition of cross-product terms to models that included main effects for alcohol and the characteristic being evaluated. All tests of statistical significance were two-sided. Statistical analyses were performed using S-PLUS (S-PLUS version 6.1 for Windows. Seattle (WA): Insightful Corporation; 2002.)Results and Discussions Table  1  summarizes the mean hormone concentrations at four weeks and eight weeks in the participants when not consuming alcohol and the percent changes from no alcohol consumption when consuming 15 g or 30 g of alcohol per day, respectively. At 4 weeks of alcohol intake the inclusion of age, years since menopause, race, and baseline BMI in the models did not change the precision of parameter estimates for alcohol doses and thus results from simple models are presented. At week 4, compared to the no alcohol placebo, estrone sulfate increased an average 6.9% (P = 0.24) when women consumed 15 g of alcohol per day and 22.2% (P = 0.0006) when they consumed 30 g of alcohol per day. DHEAS concentrations also increased significantly by an average 8.0% (P < 0.0001) on 15 g of alcohol per day and 9.2% (P < 0.0001) when 30 g alcohol was consumed per day at week 4. Trend tests for both estrone sulfate (P = 0.0006) and DHEAS (P < 0.0001) were highly significant. Table 1 Geometric mean hormone levels (ng/dL) for participants on 0 g alcohol and % change (Δ) in hormone levels from 0 g to 15 g and 30 g alcohol per day at weeks 4 and 8 Hormone 0 g/d, mean (95% C.I.) 15 g/d, Δ (95% C.I.)* 30 g/d, Δ (95% C.I.)* P -trend† Estrone Sulfate (4 wk) 47.5 (39.2–57.4) 6.9% (-4.2%–19.3%) 22.2% (9.4%–36.5%) 0.0006 (8 wk) 47.4 (40.5–55.6) 7.5% (-0.3%–15.9%) 10.7% (2.7%–19.3%) 0.009 DHEAS (4 wk) 55.3 (47.0–65.1) 8.0% (4.5%–11.6%) 9.2% (5.6%–12.8%) <0.0001 (8 wk) 59.4 (50.5–70.0) 5.1% (1.4%–9.0%) 7.5% (3.7%–11.5%) 0.0001 * Estimates of percent change are from linear mixed models, including participant as a random effect and alcohol levels as fixed effects treated as two indicator variables. †  P -trend values (two-sided) are from linear mixed models, including participant as a random effect and alcohol levels as a continuous fixed effect with values 0, 15, and 30. The effects of alcohol supplementation on serum estrone sulfate and DHEAS levels did not vary with age, BMI, race, or years since menopause. These hormone concentrations also did not differ among the three dietary periods, and the order of the assignment to the treatment regimens did not modify the associations with either of the two hormones. At 8 weeks of alcohol intake, the 15 g dose versus the placebo increased serum estrone sulfate and DHEAS by 7.5% and 5.1% respectively, whereas the 30 g dose compared to the placebo, increased levels of estrone sulfate and DHEAS by 10.7% and 7.5% respectively. When comparisons using linear mixed models are made, after controlling for alcohol intake across all doses, there was no statistically significant difference between the absolute levels of serum estrone sulfate at week 4 versus 8 (P = 0.32). However, controlling for alcohol intake absolute DHEAS levels increased between weeks 4 and 8 (P < 0.0001). The increase in DHEAS between weeks 4 and 8 did not occur in just the 15 or 30 g per day groups, however; the increase was similar in each of the three groups. In the 0 g per day group the geometric mean shifted from 55.3 ng/dL at 4 weeks to 59.4 ng/dL at 8 weeks. In the 15 g per day group the geometric mean shifted from 59.7 ng/dL to 62.5 ng/dL, and in the 30 g per day group the geometric mean shifted from 60.4 ng/dL to 63.9 ng/dL. The models did not suggest an interaction between measurement week and alcohol dose in serum estrone sulfate (P = 0.32) or DHEAS (P = 0.58). In postmenopausal women moderate alcohol consumption for four weeks resulted in statistically significant increased levels of serum estrone sulfate, the most abundant circulating estrogen, and DHEAS, the steroid hormone with the highest concentration in the blood. Compared to the placebo, serum estrone sulfate levels increased 6.9% (P = 0.24) and 22.2% (P = 0.0006) respectively among women who consumed 15 g and 30 g alcohol per day for four weeks. Serum DHEAS concentrations also increased by 8.0% (P < 0.0001) and 9.2% (P < 0.0001) respectively among women who consumed 15 g or 30 g alcohol per day for four weeks. At week 8, serum estrone sulfate levels increased 7.5% and 10.7% respectively among women who consumed 15 g and 30 g alcohol per day whereas DHEAS concentrations increased 5.1% and 7.5% respectively among women who consumed 15 g or 30 g alcohol per day compared to the placebo [ 7 ]. The increased levels of serum estrone sulfate concentrations at week 4 were essentially the same as those seen at week 8. The % change in serum estrone sulfate levels from week 4 to week 8 is -0.1%, 0.5%, and -8.9% respectively in the 0 g, 15 g, and 30 g alcohol doses. Estrone sulfate does not show a consistent change over time across all alcohol doses and there is no statistical evidence of an interaction between time and alcohol (P = 0.32). Although there appears to be a difference when looking at the point estimates in the 30 g alcohol dose, variance in estrone sulfate is so large at both 4 weeks and 8 weeks the difference between the two point estimates is not statistically significant. The large variance in estrogen sulfate at the 30 g dose will require more people to accurately judge this possible interaction. For DHEAS, the absolute levels were statistically (P < 0.0001) higher at week 8 compared to week 4 in all the alcohol groups. This increase in DHEAS between weeks 4 and 8 did not differ regardless of 0 g, 15 g, or 30 g per day dose (P = 0.58). Biological reasons may explain the increase in the 15 g and 30 g per day alcohol dose, but the reasons behind the increases in DHEAS for the 0 g dose need further research. We did not find any evidence for an effect modification between measurement week and alcohol supplementation in serum estrone sulfate (P = 0.32) or DHEAS (P = 0.58). These data indicate that the hormonal effects due to moderate consumption of alcohol equivalent to one or two drinks per day are seen early, within 4 weeks of initiation of ingestion. Importantly from a study design perspective, our study also demonstrates that it may be possible to utilize shorter study periods when assessing the effects of alcohol consumption on hormone levels, at least in postmenopausal women. To understand the earliest effects of moderate alcohol intake on hormone levels in postmenopausal women, future studies will have to be designed to assess serum levels earlier than 4 weeks or possibly later than 8 weeks. Postmenopausal women with elevated levels of serum estrone sulfate [ 8 , 9 ] and DHEAS [ 8 - 11 ] levels were reported to be at an increased risk of breast cancer in several prospective cohort studies. Results from our study showing statistically significant increased serum estrone sulfate and DHEAS concentrations after four weeks of supplementation with alcohol equivalent to one or two drinks per day, provide one possible mechanism by which moderate alcohol ingestion could increase breast cancer risk in postmenopausal women. Alcohol has many physiologic effects and could influence breast cancer risk through non-hormonal mechanisms as well. Experimental evidence suggests that alcohol interferes with folate absorption, transport, and metabolism, potentially limiting folate stores in the tissues and may interfere with DNA methylation [ 12 , 13 ]. Alcohol consumption and metabolism can result in increased production of several classes of DNA damaging molecules including reactive oxygen species [ 14 ] which can lead to increase DNA damage and the development of breast cancer [ 15 ].
Results and Discussions
Table  1  summarizes the mean hormone concentrations at four weeks and eight weeks in the participants when not consuming alcohol and the percent changes from no alcohol consumption when consuming 15 g or 30 g of alcohol per day, respectively. At 4 weeks of alcohol intake the inclusion of age, years since menopause, race, and baseline BMI in the models did not change the precision of parameter estimates for alcohol doses and thus results from simple models are presented. At week 4, compared to the no alcohol placebo, estrone sulfate increased an average 6.9% (P = 0.24) when women consumed 15 g of alcohol per day and 22.2% (P = 0.0006) when they consumed 30 g of alcohol per day. DHEAS concentrations also increased significantly by an average 8.0% (P < 0.0001) on 15 g of alcohol per day and 9.2% (P < 0.0001) when 30 g alcohol was consumed per day at week 4. Trend tests for both estrone sulfate (P = 0.0006) and DHEAS (P < 0.0001) were highly significant.Geometric mean hormone levels (ng/dL) for participants on 0 g alcohol and % change (Δ) in hormone levels from 0 g to 15 g and 30 g alcohol per day at weeks 4 and 8P* Estimates of percent change are from linear mixed models, including participant as a random effect and alcohol levels as fixed effects treated as two indicator variables.†  P -trend values (two-sided) are from linear mixed models, including participant as a random effect and alcohol levels as a continuous fixed effect with values 0, 15, and 30.PThe effects of alcohol supplementation on serum estrone sulfate and DHEAS levels did not vary with age, BMI, race, or years since menopause. These hormone concentrations also did not differ among the three dietary periods, and the order of the assignment to the treatment regimens did not modify the associations with either of the two hormones.At 8 weeks of alcohol intake, the 15 g dose versus the placebo increased serum estrone sulfate and DHEAS by 7.5% and 5.1% respectively, whereas the 30 g dose compared to the placebo, increased levels of estrone sulfate and DHEAS by 10.7% and 7.5% respectively. When comparisons using linear mixed models are made, after controlling for alcohol intake across all doses, there was no statistically significant difference between the absolute levels of serum estrone sulfate at week 4 versus 8 (P = 0.32). However, controlling for alcohol intake absolute DHEAS levels increased between weeks 4 and 8 (P < 0.0001). The increase in DHEAS between weeks 4 and 8 did not occur in just the 15 or 30 g per day groups, however; the increase was similar in each of the three groups. In the 0 g per day group the geometric mean shifted from 55.3 ng/dL at 4 weeks to 59.4 ng/dL at 8 weeks. In the 15 g per day group the geometric mean shifted from 59.7 ng/dL to 62.5 ng/dL, and in the 30 g per day group the geometric mean shifted from 60.4 ng/dL to 63.9 ng/dL. The models did not suggest an interaction between measurement week and alcohol dose in serum estrone sulfate (P = 0.32) or DHEAS (P = 0.58).In postmenopausal women moderate alcohol consumption for four weeks resulted in statistically significant increased levels of serum estrone sulfate, the most abundant circulating estrogen, and DHEAS, the steroid hormone with the highest concentration in the blood. Compared to the placebo, serum estrone sulfate levels increased 6.9% (P = 0.24) and 22.2% (P = 0.0006) respectively among women who consumed 15 g and 30 g alcohol per day for four weeks. Serum DHEAS concentrations also increased by 8.0% (P < 0.0001) and 9.2% (P < 0.0001) respectively among women who consumed 15 g or 30 g alcohol per day for four weeks. At week 8, serum estrone sulfate levels increased 7.5% and 10.7% respectively among women who consumed 15 g and 30 g alcohol per day whereas DHEAS concentrations increased 5.1% and 7.5% respectively among women who consumed 15 g or 30 g alcohol per day compared to the placebo [ 7 ]. The increased levels of serum estrone sulfate concentrations at week 4 were essentially the same as those seen at week 8. The % change in serum estrone sulfate levels from week 4 to week 8 is -0.1%, 0.5%, and -8.9% respectively in the 0 g, 15 g, and 30 g alcohol doses. Estrone sulfate does not show a consistent change over time across all alcohol doses and there is no statistical evidence of an interaction between time and alcohol (P = 0.32). Although there appears to be a difference when looking at the point estimates in the 30 g alcohol dose, variance in estrone sulfate is so large at both 4 weeks and 8 weeks the difference between the two point estimates is not statistically significant. The large variance in estrogen sulfate at the 30 g dose will require more people to accurately judge this possible interaction.For DHEAS, the absolute levels were statistically (P < 0.0001) higher at week 8 compared to week 4 in all the alcohol groups. This increase in DHEAS between weeks 4 and 8 did not differ regardless of 0 g, 15 g, or 30 g per day dose (P = 0.58). Biological reasons may explain the increase in the 15 g and 30 g per day alcohol dose, but the reasons behind the increases in DHEAS for the 0 g dose need further research. We did not find any evidence for an effect modification between measurement week and alcohol supplementation in serum estrone sulfate (P = 0.32) or DHEAS (P = 0.58).These data indicate that the hormonal effects due to moderate consumption of alcohol equivalent to one or two drinks per day are seen early, within 4 weeks of initiation of ingestion. Importantly from a study design perspective, our study also demonstrates that it may be possible to utilize shorter study periods when assessing the effects of alcohol consumption on hormone levels, at least in postmenopausal women. To understand the earliest effects of moderate alcohol intake on hormone levels in postmenopausal women, future studies will have to be designed to assess serum levels earlier than 4 weeks or possibly later than 8 weeks.Postmenopausal women with elevated levels of serum estrone sulfate [ 8 , 9 ] and DHEAS [ 8 - 11 ] levels were reported to be at an increased risk of breast cancer in several prospective cohort studies. Results from our study showing statistically significant increased serum estrone sulfate and DHEAS concentrations after four weeks of supplementation with alcohol equivalent to one or two drinks per day, provide one possible mechanism by which moderate alcohol ingestion could increase breast cancer risk in postmenopausal women.Alcohol has many physiologic effects and could influence breast cancer risk through non-hormonal mechanisms as well. Experimental evidence suggests that alcohol interferes with folate absorption, transport, and metabolism, potentially limiting folate stores in the tissues and may interfere with DNA methylation [ 12 , 13 ]. Alcohol consumption and metabolism can result in increased production of several classes of DNA damaging molecules including reactive oxygen species [ 14 ] which can lead to increase DNA damage and the development of breast cancer [ 15 ].Conclusions In conclusion, our results provide additional evidence for a mechanism by which moderate alcohol drinking could modify breast cancer risk, indicating that this effect occurs after a short time period (as early as four weeks), and thus provides further support for a causal association.
Conclusions
In conclusion, our results provide additional evidence for a mechanism by which moderate alcohol drinking could modify breast cancer risk, indicating that this effect occurs after a short time period (as early as four weeks), and thus provides further support for a causal association.Competing interests None declared.
Competing interests
None declared.


Background Colon adenocarcinomas are refractory to a number of widely used anticancer agents. Multifactorial mechanisms have been implicated in this intrinsically resistant phenotype, including deregulation of cell death pathways. In this regard, the p53 protein has a well established role in the control of tumor cell response to DNA damaging agents; however, the relationship between p53-driven genes and drug sensitivity remains controversial. The present study investigates the role of the p53/p21 system in the response of human colon carcinoma cells to treatment with the cytotoxic agent doxorubicin (DOX) and the possibility to modify the therapeutic index of DOX by modulation of p53 and/or p21 protein levels.
Background
Colon adenocarcinomas are refractory to a number of widely used anticancer agents. Multifactorial mechanisms have been implicated in this intrinsically resistant phenotype, including deregulation of cell death pathways. In this regard, the p53 protein has a well established role in the control of tumor cell response to DNA damaging agents; however, the relationship between p53-driven genes and drug sensitivity remains controversial. The present study investigates the role of the p53/p21 system in the response of human colon carcinoma cells to treatment with the cytotoxic agent doxorubicin (DOX) and the possibility to modify the therapeutic index of DOX by modulation of p53 and/or p21 protein levels.Methods The relationship between p53 and p21 protein levels and the cytotoxic effect of DOX was investigated, by MTT assay and western blot analysis, in HCT116 (p53-positive) and HT29 (p53-negative) colon cancer cells. We then assessed the effects of DOX in two isogenic cell lines derived from HCT116 by abrogating the expression and/or function of p53 and p21 (HCT116-E6 and HCT116 p21-/-, respectively). Finally, we evaluated the effect of pre-treatment with the piperidine nitroxide Tempol (TPL), an agent that was reported to induce p21 expression irrespective of p53 status, on the cytotoxicity of DOX in the four cell lines. Comparisons of IC50 values and apoptotic cell percentages were performed by ANOVA and Bonferroni's test for independent samples. C.I. calculations were performed by the combination Index method.
Methods
The relationship between p53 and p21 protein levels and the cytotoxic effect of DOX was investigated, by MTT assay and western blot analysis, in HCT116 (p53-positive) and HT29 (p53-negative) colon cancer cells. We then assessed the effects of DOX in two isogenic cell lines derived from HCT116 by abrogating the expression and/or function of p53 and p21 (HCT116-E6 and HCT116 p21-/-, respectively). Finally, we evaluated the effect of pre-treatment with the piperidine nitroxide Tempol (TPL), an agent that was reported to induce p21 expression irrespective of p53 status, on the cytotoxicity of DOX in the four cell lines. Comparisons of IC50 values and apoptotic cell percentages were performed by ANOVA and Bonferroni's test for independent samples. C.I. calculations were performed by the combination Index method.Results Our results indicate that, in the colon carcinoma cell lines tested, sensitivity to DOX is associated with p21 upregulation upon drug exposure, and DOX cytotoxicity is potentiated by pre-treatment with TPL, but only in those cell lines in which p21 can be upregulated.
Results
Our results indicate that, in the colon carcinoma cell lines tested, sensitivity to DOX is associated with p21 upregulation upon drug exposure, and DOX cytotoxicity is potentiated by pre-treatment with TPL, but only in those cell lines in which p21 can be upregulated.Conclusions p21 induction may significantly contribute to the response of colon adenocarcinomas cells to DOX treatment; and small molecules that can exploit p53-independent pathways for p21 induction, such as TPL, may find a place in chemotherapeutic protocols for the clinical management of colorectal cancer, where p53 function is often lost, due to genetic or epigenetic defects or to post-transcriptional inactivating mechanisms.
Conclusions
p21 induction may significantly contribute to the response of colon adenocarcinomas cells to DOX treatment; and small molecules that can exploit p53-independent pathways for p21 induction, such as TPL, may find a place in chemotherapeutic protocols for the clinical management of colorectal cancer, where p53 function is often lost, due to genetic or epigenetic defects or to post-transcriptional inactivating mechanisms.

Background Colorectal cancer is the second most common cause of cancer-related mortality in Western countries, with about 1 million new cases every year diagnosed world-wide and 500,000 patients dying from the disease [ 1 ]. Of the patients, 30% have advanced disease at presentation, either locally or at distant sites; in this setting, chemotherapy remains the only viable therapeutic option. However, even this option is severely hindered by the inherent resistance of metastatic colon cancer to many currently used anticancer agents. A variety of mechanisms by which cancer cells resist chemotherapy have been described, including enhanced export of drugs from cancer cells and alterations in drug metabolism and/or in drug-target interactions [ 2 ]. In addition, the response of cancer cells to genotoxic therapies may be critically impaired by defects in the response mechanisms to DNA damage [ 3 ] or in cell cycle regulatory pathways [ 4 ]. Over the past decade, induction of apoptosis has emerged as a major event in tumor cell response to cytotoxic agents (for a recent review see [ 5 ]). This view, although recently challenged by some Authors [ 6 ], has attracted considerable attention on deregulation of cell death pathways as a key determinant of drug resistance. Two separable, although extensively cross-talking, pathways leading to apoptosis have been characterized [ 7 , 8 ]. The  extrinsic pathway  is initiated by ligation of transmembrane receptors to activate membrane proximal "activator" caspases, which in turn cleave and activate downstream "effector" caspases. The  intrinsic pathway  requires disruption of the mitochondrial membrane and the release of mitochondrial proteins, two events that are regulated by the opposing actions of pro- and antiapoptotic Bcl-2 family members. "Intrinsic stresses", such as those produced by DNA-damaging agents, activate the intrinsic apoptotic pathway; the multifunctional transcription factor p53 is thought to be part of a "fast track" connection between nuclear DNA damage and the intrinsic pathway machinery [ 9 ].  p53  regulates multiple responses to genotoxic stress by transcriptional activation or repression of a number of genes encoding proteins involved in cell cycle control (p21 WAF1/Cip1 ), DNA repair (gadd45), and apoptosis (e.g. Bax, Bcl2 and survivin) [ 10 ]. Mutations in  p53  and in the p53 pathway can produce multidrug resistance  in vitro  and  in vivo , and reintroduction of wildtype  p53  into  p53  null tumor cells can re-establish chemosensitivity [ 11 ].  p53  status is not a universal predictor of treatment response, in part because not all drugs absolutely require p53 for their apoptotic function [ 12 ] and in some settings, p53 loss can enhance drug-induced apoptotic cell death [ 13 ]. Still, loss of p53 function correlates with multidrug resistance in many tumor types [ 11 ] and the observation that this is a common defect in human tumors has spurred an active search for strategies aimed at directly activating cell death pathways downstream of p53. In this scenario, the role played by the cyclin-dependent kinase inhibitor p21 is particularly intriguing, as this protein can be activated by both p53-dependent and p53-independent mechanisms and can assume pro- or anti-apoptotic functions, depending on the cellular context (for a review see [ 14 ]). The present research focuses on the role of p21 in tumor cell response to treatment with cytotoxic agents, and on the possibility to improve the therapeutic index of such agents by modulating p21 status by p53-dependent and independent pathways. The following issues have been addressed: (a) analysis of the relationship between p21 status and sensitivity to treatment with the cytotoxic anticancer agent doxorubicin (DOX) in p53-positive and -negative colon cancer cell lines; (b) design of treatment strategies based on the use of small molecules able to modulate p21 status; for the present study we have used a low molecular weight, stable nitroxide radical, 4-hydroxy-2,2,6,6,tetramethylpiperidne- N -oxyl (also known as Tempol, TPL; figure  1 ) that was shown to exert an antiproliferative effect against different cancer cell lines [ 15 ] and to increase p21 levels in a p53-null human leukemic cell line [ 16 ]. Our data indicate that p21 modulation may significantly affect cell response to DOX treatment in the colon cancer cell lines tested.
Background
Colorectal cancer is the second most common cause of cancer-related mortality in Western countries, with about 1 million new cases every year diagnosed world-wide and 500,000 patients dying from the disease [ 1 ]. Of the patients, 30% have advanced disease at presentation, either locally or at distant sites; in this setting, chemotherapy remains the only viable therapeutic option. However, even this option is severely hindered by the inherent resistance of metastatic colon cancer to many currently used anticancer agents. A variety of mechanisms by which cancer cells resist chemotherapy have been described, including enhanced export of drugs from cancer cells and alterations in drug metabolism and/or in drug-target interactions [ 2 ]. In addition, the response of cancer cells to genotoxic therapies may be critically impaired by defects in the response mechanisms to DNA damage [ 3 ] or in cell cycle regulatory pathways [ 4 ].Over the past decade, induction of apoptosis has emerged as a major event in tumor cell response to cytotoxic agents (for a recent review see [ 5 ]). This view, although recently challenged by some Authors [ 6 ], has attracted considerable attention on deregulation of cell death pathways as a key determinant of drug resistance.Two separable, although extensively cross-talking, pathways leading to apoptosis have been characterized [ 7 , 8 ]. The  extrinsic pathway  is initiated by ligation of transmembrane receptors to activate membrane proximal "activator" caspases, which in turn cleave and activate downstream "effector" caspases. The  intrinsic pathway  requires disruption of the mitochondrial membrane and the release of mitochondrial proteins, two events that are regulated by the opposing actions of pro- and antiapoptotic Bcl-2 family members. "Intrinsic stresses", such as those produced by DNA-damaging agents, activate the intrinsic apoptotic pathway; the multifunctional transcription factor p53 is thought to be part of a "fast track" connection between nuclear DNA damage and the intrinsic pathway machinery [ 9 ].  p53  regulates multiple responses to genotoxic stress by transcriptional activation or repression of a number of genes encoding proteins involved in cell cycle control (p21 WAF1/Cip1 ), DNA repair (gadd45), and apoptosis (e.g. Bax, Bcl2 and survivin) [ 10 ]. Mutations in  p53  and in the p53 pathway can produce multidrug resistance  in vitro  and  in vivo , and reintroduction of wildtype  p53  into  p53  null tumor cells can re-establish chemosensitivity [ 11 ].  p53  status is not a universal predictor of treatment response, in part because not all drugs absolutely require p53 for their apoptotic function [ 12 ] and in some settings, p53 loss can enhance drug-induced apoptotic cell death [ 13 ]. Still, loss of p53 function correlates with multidrug resistance in many tumor types [ 11 ] and the observation that this is a common defect in human tumors has spurred an active search for strategies aimed at directly activating cell death pathways downstream of p53. In this scenario, the role played by the cyclin-dependent kinase inhibitor p21 is particularly intriguing, as this protein can be activated by both p53-dependent and p53-independent mechanisms and can assume pro- or anti-apoptotic functions, depending on the cellular context (for a review see [ 14 ]).extrinsic pathwayintrinsic pathwayp53p53in vitroin vivop53p53p53The present research focuses on the role of p21 in tumor cell response to treatment with cytotoxic agents, and on the possibility to improve the therapeutic index of such agents by modulating p21 status by p53-dependent and independent pathways. The following issues have been addressed: (a) analysis of the relationship between p21 status and sensitivity to treatment with the cytotoxic anticancer agent doxorubicin (DOX) in p53-positive and -negative colon cancer cell lines; (b) design of treatment strategies based on the use of small molecules able to modulate p21 status; for the present study we have used a low molecular weight, stable nitroxide radical, 4-hydroxy-2,2,6,6,tetramethylpiperidne- N -oxyl (also known as Tempol, TPL; figure  1 ) that was shown to exert an antiproliferative effect against different cancer cell lines [ 15 ] and to increase p21 levels in a p53-null human leukemic cell line [ 16 ]. Our data indicate that p21 modulation may significantly affect cell response to DOX treatment in the colon cancer cell lines tested.NMethods Reagents Standard chemicals, including 4-hydroxy-2,2,6,6-tetramethylipiperidine- N -oxyl (Tempol, TPL) and cell culture reagents were purchased from Sigma-Aldrich srl. (Milan, Italy), unless otherwise indicated; doxorubicin (DOX) was kindly provided by Dr. A Suarato (Pfizer-Pharmacia, Milan, Italy). Cell lines The human colon carcinoma cell lines HCT116, HT29 (obtained from ATCC, Rockville, MD) and HCT116 p21-negative cells (HCT116 p21-/-), kindly provided by Dr B. Vogelstein (Johns Hopkins University, Baltimore, MD, USA), were maintained in DMEM medium supplemented with 10% FBS (Mascia Brunelli); the HCT116-E6 cell line, obtained from HCT116 cells by transfection with pCMVneo-E6 plasmid (provided by Dr B. Vogelstein) containing the HPV16-E6 human gene, was maintained in ISCOVE medium supplemented with 10% FBS and geneticin (500 μg/ml). All the cell lines were cultured at 37°C, in an atmosphere of 5% CO 2  and 95% humidity. Cytotoxicity assays The effects of DOX and/or TPL on cell growth were assessed by the MTT assay [ 17 ]. Briefly, cells were seeded onto 96-well plates and allowed to grow for 24 h prior to treatment. Three different treatment schedules were used: a) 24 h medium, 1 h DOX (0.05 – 10 μM) followed by 72 h incubation in drug-free medium; b) 24 h TPL (0.05 – 10 mM) followed by 72 h incubation in drug-free medium; c) 24 h TPL followed by 1 h DOX and by 72 h incubation in drug-free medium. For combination experiments, the whole range of DOX concentrations (0.05 – 10 μM) was tested following pretreatment with fixed TPL concentrations, corresponding to the IC 25  or IC 50 values obtained for each cell line according to schedule (b). At the end of the treatment period, 50 μl of MTT (2 mg/ml in PBS) were added to each well at 37°C for 3 h and the reduction of MTT by viable cells was measured colorimetrically at 570 nm, using a Universal Microplate Reader EL800 (Bio-Tek Instruments). IC 25  and IC 50  values (i.e. the concentrations yielding 75% and 50% cell survival fractions, respectively) were calculated according to the median effect equation and analysis of the interaction between DOX and TPL was performed as described by Chou & Talalay [ 18 ]. Transfection of HCT116 cells Transfection of HCT116 cells with the pCMVneo-E6 plasmid was performed by electroporation as described by Yanez and Porter [ 19 ], using a Bio-Rad Gene Pulser unit at the following conditions: 280 V, 960 μF. 48 h post-transfection the cells were selected by adding 500 μg/ml of geneticin to the culture medium. The efficiency and stability of transfection were checked by Western blot analysis of whole cell lysates. Control cells were mock-transfected with the pCMVneo plasmid. Preparation of cell extracts and immunoblotting The expression of p53 and p21, before and after 1 h exposure to DOX (1.0 and 10 μM) followed by 23 h incubation in drug-free medium, or after 24 h exposure to TPL (1.0 and 2.5 mM), was evaluated by Western blot analysis of total protein extracts (lysis buffer: NP40 1%, leupeptin 10 μg/ml and aprotinin 10 μg/ml in TBS). Protein concentration in the cellular lysates was determined by the BCA assay (Pierce, Rockford, IL, USA). 15 μg of protein extract/lane were loaded onto 11% polyacrylamide gels and separated under denaturing conditions. Protein samples were then transferred onto nitrocellulose membrane and Western blot analysis was performed by standard techniques. using a mouse anti-p53 monoclonal antibody (DO-1; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and a rabbit anti-p21 polyclonal antibody (C-19; Santa Cruz Biotechnology). Proteins were visualized using peroxidase-conjugated anti-mouse and anti-rabbit secondary antibodies (Amersham Pharmacia Biotech) and the ECL Plus Western Blotting Detection Reagents (Amersham Pharmacia Biotech). Densitometric analysis was performed using the Scion Image software (Scion Corporation, Frederick, MD). Flow cytometric analysis of apoptotic cells The presence of apoptotic cells in HCT116, HT29, HCT116-E6 and HCT116 p21 -/-, before and after 1 h exposure to DOX (1.0 and 10 μM) followed by 23 h incubation in drug-free medium, was evaluated by flow cytometric analysis, using a Becton Dickinson FACScalibur flow cytometer. Cells were detached by trypsinization, washed in phosphate-buffered saline (PBS) and fixed in ice-cold 70% ethanol for 20 min at -20°C. After an additional wash in PBS, DNA was stained with 50 mg/ml propidium iodide in PBS in the presence of RNAse A (30 U/ml) at 37°C for 30 minutes. 5 × 10 5  cell samples were analyzed and data were processed using the CellQuest software (Becton Dickinson). The percentage of apoptotic cells in each sample was determined based on the sub-G1 peaks detected in monoparametric histograms. Statistical analysis Comparisons of IC 50  values and apoptotic cell percentages in the four different cell lines were performed by ANOVA and Bonferroni's test for independent samples. C.I. calculations an relative statistical analysis were performed as described by Chou and Talalay [ 18 ]. According to this method, a  combination index  (C.I.) can be calculated from dose-response curves obtained following exposure to DOX and/or TPL as single agents and in combination. C.I. values approximating 1.0 indicate additive interactions between the two agents; C.I. < 1.0 indicate synergy and, conversely, C.I. > 1.0 indicate antagonism.
Methods
Reagents Standard chemicals, including 4-hydroxy-2,2,6,6-tetramethylipiperidine- N -oxyl (Tempol, TPL) and cell culture reagents were purchased from Sigma-Aldrich srl. (Milan, Italy), unless otherwise indicated; doxorubicin (DOX) was kindly provided by Dr. A Suarato (Pfizer-Pharmacia, Milan, Italy).
Reagents
Standard chemicals, including 4-hydroxy-2,2,6,6-tetramethylipiperidine- N -oxyl (Tempol, TPL) and cell culture reagents were purchased from Sigma-Aldrich srl. (Milan, Italy), unless otherwise indicated; doxorubicin (DOX) was kindly provided by Dr. A Suarato (Pfizer-Pharmacia, Milan, Italy).NCell lines The human colon carcinoma cell lines HCT116, HT29 (obtained from ATCC, Rockville, MD) and HCT116 p21-negative cells (HCT116 p21-/-), kindly provided by Dr B. Vogelstein (Johns Hopkins University, Baltimore, MD, USA), were maintained in DMEM medium supplemented with 10% FBS (Mascia Brunelli); the HCT116-E6 cell line, obtained from HCT116 cells by transfection with pCMVneo-E6 plasmid (provided by Dr B. Vogelstein) containing the HPV16-E6 human gene, was maintained in ISCOVE medium supplemented with 10% FBS and geneticin (500 μg/ml). All the cell lines were cultured at 37°C, in an atmosphere of 5% CO 2  and 95% humidity.
Cell lines
The human colon carcinoma cell lines HCT116, HT29 (obtained from ATCC, Rockville, MD) and HCT116 p21-negative cells (HCT116 p21-/-), kindly provided by Dr B. Vogelstein (Johns Hopkins University, Baltimore, MD, USA), were maintained in DMEM medium supplemented with 10% FBS (Mascia Brunelli); the HCT116-E6 cell line, obtained from HCT116 cells by transfection with pCMVneo-E6 plasmid (provided by Dr B. Vogelstein) containing the HPV16-E6 human gene, was maintained in ISCOVE medium supplemented with 10% FBS and geneticin (500 μg/ml). All the cell lines were cultured at 37°C, in an atmosphere of 5% CO 2  and 95% humidity.Cytotoxicity assays The effects of DOX and/or TPL on cell growth were assessed by the MTT assay [ 17 ]. Briefly, cells were seeded onto 96-well plates and allowed to grow for 24 h prior to treatment. Three different treatment schedules were used: a) 24 h medium, 1 h DOX (0.05 – 10 μM) followed by 72 h incubation in drug-free medium; b) 24 h TPL (0.05 – 10 mM) followed by 72 h incubation in drug-free medium; c) 24 h TPL followed by 1 h DOX and by 72 h incubation in drug-free medium. For combination experiments, the whole range of DOX concentrations (0.05 – 10 μM) was tested following pretreatment with fixed TPL concentrations, corresponding to the IC 25  or IC 50 values obtained for each cell line according to schedule (b). At the end of the treatment period, 50 μl of MTT (2 mg/ml in PBS) were added to each well at 37°C for 3 h and the reduction of MTT by viable cells was measured colorimetrically at 570 nm, using a Universal Microplate Reader EL800 (Bio-Tek Instruments). IC 25  and IC 50  values (i.e. the concentrations yielding 75% and 50% cell survival fractions, respectively) were calculated according to the median effect equation and analysis of the interaction between DOX and TPL was performed as described by Chou & Talalay [ 18 ].
Cytotoxicity assays
The effects of DOX and/or TPL on cell growth were assessed by the MTT assay [ 17 ]. Briefly, cells were seeded onto 96-well plates and allowed to grow for 24 h prior to treatment. Three different treatment schedules were used: a) 24 h medium, 1 h DOX (0.05 – 10 μM) followed by 72 h incubation in drug-free medium; b) 24 h TPL (0.05 – 10 mM) followed by 72 h incubation in drug-free medium; c) 24 h TPL followed by 1 h DOX and by 72 h incubation in drug-free medium. For combination experiments, the whole range of DOX concentrations (0.05 – 10 μM) was tested following pretreatment with fixed TPL concentrations, corresponding to the IC 25  or IC 50 values obtained for each cell line according to schedule (b). At the end of the treatment period, 50 μl of MTT (2 mg/ml in PBS) were added to each well at 37°C for 3 h and the reduction of MTT by viable cells was measured colorimetrically at 570 nm, using a Universal Microplate Reader EL800 (Bio-Tek Instruments). IC 25  and IC 50  values (i.e. the concentrations yielding 75% and 50% cell survival fractions, respectively) were calculated according to the median effect equation and analysis of the interaction between DOX and TPL was performed as described by Chou & Talalay [ 18 ].Transfection of HCT116 cells Transfection of HCT116 cells with the pCMVneo-E6 plasmid was performed by electroporation as described by Yanez and Porter [ 19 ], using a Bio-Rad Gene Pulser unit at the following conditions: 280 V, 960 μF. 48 h post-transfection the cells were selected by adding 500 μg/ml of geneticin to the culture medium. The efficiency and stability of transfection were checked by Western blot analysis of whole cell lysates. Control cells were mock-transfected with the pCMVneo plasmid.
Transfection of HCT116 cells
Transfection of HCT116 cells with the pCMVneo-E6 plasmid was performed by electroporation as described by Yanez and Porter [ 19 ], using a Bio-Rad Gene Pulser unit at the following conditions: 280 V, 960 μF. 48 h post-transfection the cells were selected by adding 500 μg/ml of geneticin to the culture medium. The efficiency and stability of transfection were checked by Western blot analysis of whole cell lysates. Control cells were mock-transfected with the pCMVneo plasmid.Preparation of cell extracts and immunoblotting The expression of p53 and p21, before and after 1 h exposure to DOX (1.0 and 10 μM) followed by 23 h incubation in drug-free medium, or after 24 h exposure to TPL (1.0 and 2.5 mM), was evaluated by Western blot analysis of total protein extracts (lysis buffer: NP40 1%, leupeptin 10 μg/ml and aprotinin 10 μg/ml in TBS). Protein concentration in the cellular lysates was determined by the BCA assay (Pierce, Rockford, IL, USA). 15 μg of protein extract/lane were loaded onto 11% polyacrylamide gels and separated under denaturing conditions. Protein samples were then transferred onto nitrocellulose membrane and Western blot analysis was performed by standard techniques. using a mouse anti-p53 monoclonal antibody (DO-1; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and a rabbit anti-p21 polyclonal antibody (C-19; Santa Cruz Biotechnology). Proteins were visualized using peroxidase-conjugated anti-mouse and anti-rabbit secondary antibodies (Amersham Pharmacia Biotech) and the ECL Plus Western Blotting Detection Reagents (Amersham Pharmacia Biotech). Densitometric analysis was performed using the Scion Image software (Scion Corporation, Frederick, MD).
Preparation of cell extracts and immunoblotting
The expression of p53 and p21, before and after 1 h exposure to DOX (1.0 and 10 μM) followed by 23 h incubation in drug-free medium, or after 24 h exposure to TPL (1.0 and 2.5 mM), was evaluated by Western blot analysis of total protein extracts (lysis buffer: NP40 1%, leupeptin 10 μg/ml and aprotinin 10 μg/ml in TBS). Protein concentration in the cellular lysates was determined by the BCA assay (Pierce, Rockford, IL, USA). 15 μg of protein extract/lane were loaded onto 11% polyacrylamide gels and separated under denaturing conditions. Protein samples were then transferred onto nitrocellulose membrane and Western blot analysis was performed by standard techniques. using a mouse anti-p53 monoclonal antibody (DO-1; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and a rabbit anti-p21 polyclonal antibody (C-19; Santa Cruz Biotechnology). Proteins were visualized using peroxidase-conjugated anti-mouse and anti-rabbit secondary antibodies (Amersham Pharmacia Biotech) and the ECL Plus Western Blotting Detection Reagents (Amersham Pharmacia Biotech).Densitometric analysis was performed using the Scion Image software (Scion Corporation, Frederick, MD).Flow cytometric analysis of apoptotic cells The presence of apoptotic cells in HCT116, HT29, HCT116-E6 and HCT116 p21 -/-, before and after 1 h exposure to DOX (1.0 and 10 μM) followed by 23 h incubation in drug-free medium, was evaluated by flow cytometric analysis, using a Becton Dickinson FACScalibur flow cytometer. Cells were detached by trypsinization, washed in phosphate-buffered saline (PBS) and fixed in ice-cold 70% ethanol for 20 min at -20°C. After an additional wash in PBS, DNA was stained with 50 mg/ml propidium iodide in PBS in the presence of RNAse A (30 U/ml) at 37°C for 30 minutes. 5 × 10 5  cell samples were analyzed and data were processed using the CellQuest software (Becton Dickinson). The percentage of apoptotic cells in each sample was determined based on the sub-G1 peaks detected in monoparametric histograms.
Flow cytometric analysis of apoptotic cells
The presence of apoptotic cells in HCT116, HT29, HCT116-E6 and HCT116 p21 -/-, before and after 1 h exposure to DOX (1.0 and 10 μM) followed by 23 h incubation in drug-free medium, was evaluated by flow cytometric analysis, using a Becton Dickinson FACScalibur flow cytometer. Cells were detached by trypsinization, washed in phosphate-buffered saline (PBS) and fixed in ice-cold 70% ethanol for 20 min at -20°C. After an additional wash in PBS, DNA was stained with 50 mg/ml propidium iodide in PBS in the presence of RNAse A (30 U/ml) at 37°C for 30 minutes. 5 × 10 5  cell samples were analyzed and data were processed using the CellQuest software (Becton Dickinson). The percentage of apoptotic cells in each sample was determined based on the sub-G1 peaks detected in monoparametric histograms.Statistical analysis Comparisons of IC 50  values and apoptotic cell percentages in the four different cell lines were performed by ANOVA and Bonferroni's test for independent samples. C.I. calculations an relative statistical analysis were performed as described by Chou and Talalay [ 18 ]. According to this method, a  combination index  (C.I.) can be calculated from dose-response curves obtained following exposure to DOX and/or TPL as single agents and in combination. C.I. values approximating 1.0 indicate additive interactions between the two agents; C.I. < 1.0 indicate synergy and, conversely, C.I. > 1.0 indicate antagonism.
Statistical analysis
Comparisons of IC 50  values and apoptotic cell percentages in the four different cell lines were performed by ANOVA and Bonferroni's test for independent samples. C.I. calculations an relative statistical analysis were performed as described by Chou and Talalay [ 18 ]. According to this method, a  combination index  (C.I.) can be calculated from dose-response curves obtained following exposure to DOX and/or TPL as single agents and in combination. C.I. values approximating 1.0 indicate additive interactions between the two agents; C.I. < 1.0 indicate synergy and, conversely, C.I. > 1.0 indicate antagonism.combination indexResults Cytotoxicity assays Figure  2  shows the dose-response curves for HCT116, HT29, HCT116-E6 and HCT116 p21-/- cells following 24 h incubation in drug-free medium, 1 h exposure to DOX and 72 h incubation in drug-free medium. IC 50  values calculated from these data are reported in table  1 ; the  resistance index  (R.I.) is calculated as the ratio between the IC 50  value obtained for each cell line and that obtained for HCT116 cells. The HT29 cell line, which carries a mutant form of the  p53  gene, is significantly more resistant to the cytotoxic action of DOX than the HCT116 cell line, carrying a wild-type  p53  gene (IC 50  values: 2.197 ± 0.11 μM vs. 0.38 ± 0.03 μM, respectively; mean ± s.e.m. of 4–6 experiments,  p  < 0.05). HCT116-E6 cells are 2-fold more resistant to DOX than HCT116 cells (IC 50 : 0.770 ± 0.06 μM; mean ± s.e.m. of 4–6 experiments,  p  < 0.05 vs. HCT116), while HCT116 p21-/- cells are 14-fold more resistant to DOX than HCT116 cell line (IC 50 : 5.457 ± 0.163 μM; mean ± s.e.m. of 4–6 experiments,  p  < 0.05 vs. HCT116), in spite of the presence of a wild type form of p53. Immune detection of p53 and p21 Figure  3  shows the expression of p53 (A) and p21 (C) proteins, before and after exposure to DOX (1 and 10  M for 1 h followed by 23 h in drug-free medium), in the four cell lines tested; panels B and D report the densitometric analysis of p53 and p21 immune reactive bands, respectively. In the absence of drug treatment, it is possible to observe that HT29 cells show higher p53 but lower p21 protein levels, as compared with the HCT116 cell line. DOX treatment induces a dose-dependent increase in both p53 and p21 levels in HCT116 cells, whereas in HT29 cells p53 protein levels are not significantly modified by the treatment and p21 protein levels are only detectable when 25 μg of protein extract/lane are loaded onto the gel (instead of the 15 μg loaded for the other cell lines), and even then only at the highest DOX concentration used. As expected, HCT116 E6 cells do not show detectable p53 levels, both under baseline conditions and following DOX treatment; in contrast in HCT116-E6 cells p21 levels are increased by DOX treatment in a dose-dependent fashion, although to a lesser extent than in HCT116 cells. HCT116 p21-/- cells show higher baseline expression levels of p53 compared with HCT116 cells and DOX treatment in this cell line enhances p53 expression to a an even greater extent than in HCT116 cells line. As expected, p21 is undetectable in HCT116 p21-/- cells, and DOX treatment does not modify the intracellular levels of this protein. Evaluation of apoptotic cells by flow cytometric analysis Figure  4  shows the percentage of apoptotic cells following treatment of the four colon cell lines with DOX (1.0 and 10 μM) for 1 h followed by 23 h in drug-free medium. No significant differences in the percentage of apoptotic cells were observed in untreated HCT116, HT29, HCT116-E6 and HCT116 p21-/- cells. Exposure to DOX induces concentration-dependent increases in apoptotic cells in all the cell lines tested; HCT116-E6 cells were the least susceptible apoptosis induction by DOX. Effects of TPL on cell survival and p53/p21 levels Table  2  reports the IC 25  and IC 50  values obtained for the four cell lines after 24 h of continuous TPL exposure followed by 72 h in drug-free medium. TPL can be observed to inhibit cell growth in all four cell lines; although no significant differences can be detected among IC 50  values, HCT116 p21-/- cells appear to be less responsive than the other three cell lines. Figure  5  shows that 24 h exposure to TPL (1 and 2.5 mM) induces a dose-dependent increase in both p53 and in p21 levels in HCT116 cells, whereas in HT29 cell line TPL treatment only induces a dose-dependent increase in p21 expression. Exposure of HCT116-E6 cells to TPL (1 and 2.5 mM) for 24 h does not induce any variations in p53 expression, while a dose-dependent increase in p21 expression can be observed following treatment with the nitroxide. In HCT116 p21-/- cells TPL induce a slight increase in p53 protein levels but, as expected, p21 levels were unaffected by TPL treatment. Effects of TPL pretreatment on DOX-induced cytotoxicity Figure  6  shows the effect of 24 h pretreatment with TPL, at fixed concentrations corresponding to the IC 25  and IC 50  values obtained for each cell line, on DOX cytotoxicity. The cells' response to DOX is expressed as the IC 50  values derived from dose/response curves obtained after 1 h exposure to DOX with or without pretreatment with TPL (24 h), followed by 72 h in drug-free medium. Analysis of cytotoxicity data shows a synergistic interaction (C.I.<1) between DOX and TPL for both TPL concentrations in HCT116 cells and in HCT116-E6 and HT29 cells at the lower concentration; only additive effects (C.I. ≈ 1) can be observed in HCT116 p21 -/- cells. IC 50  values for DOX and TPL according to the three different schedules are reported in table  3 .
Results
Cytotoxicity assays Figure  2  shows the dose-response curves for HCT116, HT29, HCT116-E6 and HCT116 p21-/- cells following 24 h incubation in drug-free medium, 1 h exposure to DOX and 72 h incubation in drug-free medium. IC 50  values calculated from these data are reported in table  1 ; the  resistance index  (R.I.) is calculated as the ratio between the IC 50  value obtained for each cell line and that obtained for HCT116 cells. The HT29 cell line, which carries a mutant form of the  p53  gene, is significantly more resistant to the cytotoxic action of DOX than the HCT116 cell line, carrying a wild-type  p53  gene (IC 50  values: 2.197 ± 0.11 μM vs. 0.38 ± 0.03 μM, respectively; mean ± s.e.m. of 4–6 experiments,  p  < 0.05). HCT116-E6 cells are 2-fold more resistant to DOX than HCT116 cells (IC 50 : 0.770 ± 0.06 μM; mean ± s.e.m. of 4–6 experiments,  p  < 0.05 vs. HCT116), while HCT116 p21-/- cells are 14-fold more resistant to DOX than HCT116 cell line (IC 50 : 5.457 ± 0.163 μM; mean ± s.e.m. of 4–6 experiments,  p  < 0.05 vs. HCT116), in spite of the presence of a wild type form of p53.
Cytotoxicity assays
Figure  2  shows the dose-response curves for HCT116, HT29, HCT116-E6 and HCT116 p21-/- cells following 24 h incubation in drug-free medium, 1 h exposure to DOX and 72 h incubation in drug-free medium. IC 50  values calculated from these data are reported in table  1 ; the  resistance index  (R.I.) is calculated as the ratio between the IC 50  value obtained for each cell line and that obtained for HCT116 cells. The HT29 cell line, which carries a mutant form of the  p53  gene, is significantly more resistant to the cytotoxic action of DOX than the HCT116 cell line, carrying a wild-type  p53  gene (IC 50  values: 2.197 ± 0.11 μM vs. 0.38 ± 0.03 μM, respectively; mean ± s.e.m. of 4–6 experiments,  p  < 0.05). HCT116-E6 cells are 2-fold more resistant to DOX than HCT116 cells (IC 50 : 0.770 ± 0.06 μM; mean ± s.e.m. of 4–6 experiments,  p  < 0.05 vs. HCT116), while HCT116 p21-/- cells are 14-fold more resistant to DOX than HCT116 cell line (IC 50 : 5.457 ± 0.163 μM; mean ± s.e.m. of 4–6 experiments,  p  < 0.05 vs. HCT116), in spite of the presence of a wild type form of p53.resistance indexp53p53pppImmune detection of p53 and p21 Figure  3  shows the expression of p53 (A) and p21 (C) proteins, before and after exposure to DOX (1 and 10  M for 1 h followed by 23 h in drug-free medium), in the four cell lines tested; panels B and D report the densitometric analysis of p53 and p21 immune reactive bands, respectively. In the absence of drug treatment, it is possible to observe that HT29 cells show higher p53 but lower p21 protein levels, as compared with the HCT116 cell line. DOX treatment induces a dose-dependent increase in both p53 and p21 levels in HCT116 cells, whereas in HT29 cells p53 protein levels are not significantly modified by the treatment and p21 protein levels are only detectable when 25 μg of protein extract/lane are loaded onto the gel (instead of the 15 μg loaded for the other cell lines), and even then only at the highest DOX concentration used. As expected, HCT116 E6 cells do not show detectable p53 levels, both under baseline conditions and following DOX treatment; in contrast in HCT116-E6 cells p21 levels are increased by DOX treatment in a dose-dependent fashion, although to a lesser extent than in HCT116 cells. HCT116 p21-/- cells show higher baseline expression levels of p53 compared with HCT116 cells and DOX treatment in this cell line enhances p53 expression to a an even greater extent than in HCT116 cells line. As expected, p21 is undetectable in HCT116 p21-/- cells, and DOX treatment does not modify the intracellular levels of this protein.
Immune detection of p53 and p21
Figure  3  shows the expression of p53 (A) and p21 (C) proteins, before and after exposure to DOX (1 and 10  M for 1 h followed by 23 h in drug-free medium), in the four cell lines tested; panels B and D report the densitometric analysis of p53 and p21 immune reactive bands, respectively. In the absence of drug treatment, it is possible to observe that HT29 cells show higher p53 but lower p21 protein levels, as compared with the HCT116 cell line. DOX treatment induces a dose-dependent increase in both p53 and p21 levels in HCT116 cells, whereas in HT29 cells p53 protein levels are not significantly modified by the treatment and p21 protein levels are only detectable when 25 μg of protein extract/lane are loaded onto the gel (instead of the 15 μg loaded for the other cell lines), and even then only at the highest DOX concentration used. As expected, HCT116 E6 cells do not show detectable p53 levels, both under baseline conditions and following DOX treatment; in contrast in HCT116-E6 cells p21 levels are increased by DOX treatment in a dose-dependent fashion, although to a lesser extent than in HCT116 cells. HCT116 p21-/- cells show higher baseline expression levels of p53 compared with HCT116 cells and DOX treatment in this cell line enhances p53 expression to a an even greater extent than in HCT116 cells line. As expected, p21 is undetectable in HCT116 p21-/- cells, and DOX treatment does not modify the intracellular levels of this protein.Evaluation of apoptotic cells by flow cytometric analysis Figure  4  shows the percentage of apoptotic cells following treatment of the four colon cell lines with DOX (1.0 and 10 μM) for 1 h followed by 23 h in drug-free medium. No significant differences in the percentage of apoptotic cells were observed in untreated HCT116, HT29, HCT116-E6 and HCT116 p21-/- cells. Exposure to DOX induces concentration-dependent increases in apoptotic cells in all the cell lines tested; HCT116-E6 cells were the least susceptible apoptosis induction by DOX.
Evaluation of apoptotic cells by flow cytometric analysis
Figure  4  shows the percentage of apoptotic cells following treatment of the four colon cell lines with DOX (1.0 and 10 μM) for 1 h followed by 23 h in drug-free medium. No significant differences in the percentage of apoptotic cells were observed in untreated HCT116, HT29, HCT116-E6 and HCT116 p21-/- cells. Exposure to DOX induces concentration-dependent increases in apoptotic cells in all the cell lines tested; HCT116-E6 cells were the least susceptible apoptosis induction by DOX.Effects of TPL on cell survival and p53/p21 levels Table  2  reports the IC 25  and IC 50  values obtained for the four cell lines after 24 h of continuous TPL exposure followed by 72 h in drug-free medium. TPL can be observed to inhibit cell growth in all four cell lines; although no significant differences can be detected among IC 50  values, HCT116 p21-/- cells appear to be less responsive than the other three cell lines. Figure  5  shows that 24 h exposure to TPL (1 and 2.5 mM) induces a dose-dependent increase in both p53 and in p21 levels in HCT116 cells, whereas in HT29 cell line TPL treatment only induces a dose-dependent increase in p21 expression. Exposure of HCT116-E6 cells to TPL (1 and 2.5 mM) for 24 h does not induce any variations in p53 expression, while a dose-dependent increase in p21 expression can be observed following treatment with the nitroxide. In HCT116 p21-/- cells TPL induce a slight increase in p53 protein levels but, as expected, p21 levels were unaffected by TPL treatment.
Effects of TPL on cell survival and p53/p21 levels
Table  2  reports the IC 25  and IC 50  values obtained for the four cell lines after 24 h of continuous TPL exposure followed by 72 h in drug-free medium. TPL can be observed to inhibit cell growth in all four cell lines; although no significant differences can be detected among IC 50  values, HCT116 p21-/- cells appear to be less responsive than the other three cell lines. Figure  5  shows that 24 h exposure to TPL (1 and 2.5 mM) induces a dose-dependent increase in both p53 and in p21 levels in HCT116 cells, whereas in HT29 cell line TPL treatment only induces a dose-dependent increase in p21 expression. Exposure of HCT116-E6 cells to TPL (1 and 2.5 mM) for 24 h does not induce any variations in p53 expression, while a dose-dependent increase in p21 expression can be observed following treatment with the nitroxide. In HCT116 p21-/- cells TPL induce a slight increase in p53 protein levels but, as expected, p21 levels were unaffected by TPL treatment.Effects of TPL pretreatment on DOX-induced cytotoxicity Figure  6  shows the effect of 24 h pretreatment with TPL, at fixed concentrations corresponding to the IC 25  and IC 50  values obtained for each cell line, on DOX cytotoxicity. The cells' response to DOX is expressed as the IC 50  values derived from dose/response curves obtained after 1 h exposure to DOX with or without pretreatment with TPL (24 h), followed by 72 h in drug-free medium. Analysis of cytotoxicity data shows a synergistic interaction (C.I.<1) between DOX and TPL for both TPL concentrations in HCT116 cells and in HCT116-E6 and HT29 cells at the lower concentration; only additive effects (C.I. ≈ 1) can be observed in HCT116 p21 -/- cells. IC 50  values for DOX and TPL according to the three different schedules are reported in table  3 .
Effects of TPL pretreatment on DOX-induced cytotoxicity
Figure  6  shows the effect of 24 h pretreatment with TPL, at fixed concentrations corresponding to the IC 25  and IC 50  values obtained for each cell line, on DOX cytotoxicity. The cells' response to DOX is expressed as the IC 50  values derived from dose/response curves obtained after 1 h exposure to DOX with or without pretreatment with TPL (24 h), followed by 72 h in drug-free medium. Analysis of cytotoxicity data shows a synergistic interaction (C.I.<1) between DOX and TPL for both TPL concentrations in HCT116 cells and in HCT116-E6 and HT29 cells at the lower concentration; only additive effects (C.I. ≈ 1) can be observed in HCT116 p21 -/- cells. IC 50  values for DOX and TPL according to the three different schedules are reported in table  3 .Discussion Resistance of colorectal cancer to established treatment regimens remains a major concern in oncology; thus attempts at improving the survival of patients affected by this disease depend largely on strategies targeting tumor cell resistance, which cannot be rationally planned without a detailed knowledge of the mechanisms underlying this phenomenon. A current paradigm regarding cancer chemotherapy indicates disabling of the intrinsic apoptotic pathway as a key factor in the response of tumor cells to anticancer drugs [ 3 , 5 , 12 ]. Therefore, strategies aiming at re-establishing the cell's capability to activate a cell death program are an area of active research. The present study was performed in order to define the role of the p53/p21 pathway in the response of colorectal carcinoma cells to DOX, a cytotoxic agent that is typically devoid of effects in this tumor type. The results obtained in our cytotoxicity studies indicate that in the cell lines examined  p53  status is not unequivocally related to the response to DOX: in fact, while  p53 -deficient cells (HT-29, HCT116-E6) are indeed less responsive than the  p53/wt  parental HCT116 cell line, the highest resistance index was obtained for HCT116 p21-/- cells, harboring two wildtype  p53  alleles. As expected, treatment with DOX leads to p53 upregulation in the cell lines expressing wildtype p53; this effect has been thoroughly documented in colon cancer cells as well as in tumor cell lines derived from other tissues, and has been attributed to phosphorylation and subsequent stabilization of p53, possibly through activation of DNA-dependent protein kinase or ATM (ataxia-teleangectasia mutated) kinase (see e.g. [ 20 - 22 ]). In HCT116 cells, p21 expression parallels p53 activation; however, data obtained in HT29 and HCT116-E6 cells clearly indicate the existence of p53-independent pathways for p21 induction, that have been extensively characterized (for a review see [ 23 ]) and can be activated to variable extents (HCT116 E6 > HT29) upon exposure to DOX. Interestingly, the extent of the cytotoxic effects observed in the small panel of colon cell lines tested rather seems to parallel the cells' ability to upregulate p21 (HCT116 > HCT116-E6 > HT29 > HCT116 p21-/-). This result is somewhat unexpected: in fact, whereas the function of p21 in cell growth arrest following DNA damage has been established for a long time [ 24 ], the role played by this protein in the ultimate fate of tumor cells exposed to cytotoxic agents is far from clear-cut [ 14 , 25 ]. In a number of studies, p21 has actually been reported to protect tumor cells against cell death induced by enforced p53 expression [ 26 ] or by low doses of cytotoxic agents [ 13 , 27 - 30 ]. However, in other experimental settings, p53-dependent or -independent induction of p21 expression seems to be a prerequisite for apoptosis [ 31 - 34 ] and to sensitize tumor cells to the action of different agents [ 35 - 37 ]. The putative mechanisms by which p21 might actually induce apoptosis have recently been reviewed [ 38 ], but still await full elucidation. Interestingly, the situation outlined by our results does not seem to conform to either view: in fact, while induction of p21 in p53-proficient and -deficient cell lines is associated with increased response to drug treatment, this was not accompanied by a parallel increase in apoptotic cells, as no significant differences in apoptosis were observed between HCT116 cells and the 14-fold resistant HCT-116 p21-/- cell line (figure  4 ). This suggests that modes of cell death other than apoptosis may operate in tumor cells following exposure to DOX, or, more generally, to DNA-damaging agents, a concept that is beginning to be proposed by a number of Authors [ 39 , 40 ]; of note, recent experimental evidence indicates p21 as one of the major determinants of terminal growth arrest induced by cytotoxic agents [ 41 - 43 ]. Therefore, although issues related to terminal growth arrest and senescence have not been specifically addressed in the present study, the possibility that these phenomena might play a role linking cell death to the observed increases in p21 levels should not be disregarded. The hypothesis that the cytotoxic response of the tumor cell lines tested in the present study may depend on p21 induction is further corroborated by data obtained following pre-treatment with the piperidine nitroxide TPL. The choice of this compound was dictated by previous findings indicating that TPL induces cell death in a number of tumor cell lines irrespective of their  p53  status [ 15 ], and that it increases p21 levels in  p53 -null cells [ 16 ]. The results of the present study show that TPL affects the four colon cell lines to similar extents, thus confirming that its growth inhibitory effect is independent of p53 function. HCT-116 p21-/- cells are actually slightly less responsive than the other cell lines [even though the difference does not attain statistical significance), which suggests the possibility that the effects of TPL are due in part to its ability to increase p21 levels. Interestingly, the nitroxide also induces p21 expression even in p53-deficient cell lines; this observation suggests that TPL can activate p53-independent pathways for p21 induction, as already noted following exposure of HT29 and HCT116 E6 cells to DOX. Moreover, activation of such pathways by TPL appears to sensitize tumor cells to the action of DOX: in fact, synergistic potentiation of DOX cytotoxicity is achieved by TPL in those cell lines where p21 expression can be induced, but only additive effects between TPL and DOX are observed in HCT116 p21-/-, where p21 expression is constitutively absent.
Discussion
Resistance of colorectal cancer to established treatment regimens remains a major concern in oncology; thus attempts at improving the survival of patients affected by this disease depend largely on strategies targeting tumor cell resistance, which cannot be rationally planned without a detailed knowledge of the mechanisms underlying this phenomenon. A current paradigm regarding cancer chemotherapy indicates disabling of the intrinsic apoptotic pathway as a key factor in the response of tumor cells to anticancer drugs [ 3 , 5 , 12 ]. Therefore, strategies aiming at re-establishing the cell's capability to activate a cell death program are an area of active research. The present study was performed in order to define the role of the p53/p21 pathway in the response of colorectal carcinoma cells to DOX, a cytotoxic agent that is typically devoid of effects in this tumor type. The results obtained in our cytotoxicity studies indicate that in the cell lines examined  p53  status is not unequivocally related to the response to DOX: in fact, while  p53 -deficient cells (HT-29, HCT116-E6) are indeed less responsive than the  p53/wt  parental HCT116 cell line, the highest resistance index was obtained for HCT116 p21-/- cells, harboring two wildtype  p53  alleles. As expected, treatment with DOX leads to p53 upregulation in the cell lines expressing wildtype p53; this effect has been thoroughly documented in colon cancer cells as well as in tumor cell lines derived from other tissues, and has been attributed to phosphorylation and subsequent stabilization of p53, possibly through activation of DNA-dependent protein kinase or ATM (ataxia-teleangectasia mutated) kinase (see e.g. [ 20 - 22 ]). In HCT116 cells, p21 expression parallels p53 activation; however, data obtained in HT29 and HCT116-E6 cells clearly indicate the existence of p53-independent pathways for p21 induction, that have been extensively characterized (for a review see [ 23 ]) and can be activated to variable extents (HCT116 E6 > HT29) upon exposure to DOX. Interestingly, the extent of the cytotoxic effects observed in the small panel of colon cell lines tested rather seems to parallel the cells' ability to upregulate p21 (HCT116 > HCT116-E6 > HT29 > HCT116 p21-/-). This result is somewhat unexpected: in fact, whereas the function of p21 in cell growth arrest following DNA damage has been established for a long time [ 24 ], the role played by this protein in the ultimate fate of tumor cells exposed to cytotoxic agents is far from clear-cut [ 14 , 25 ]. In a number of studies, p21 has actually been reported to protect tumor cells against cell death induced by enforced p53 expression [ 26 ] or by low doses of cytotoxic agents [ 13 , 27 - 30 ]. However, in other experimental settings, p53-dependent or -independent induction of p21 expression seems to be a prerequisite for apoptosis [ 31 - 34 ] and to sensitize tumor cells to the action of different agents [ 35 - 37 ]. The putative mechanisms by which p21 might actually induce apoptosis have recently been reviewed [ 38 ], but still await full elucidation.p53p53p53/wtp53Interestingly, the situation outlined by our results does not seem to conform to either view: in fact, while induction of p21 in p53-proficient and -deficient cell lines is associated with increased response to drug treatment, this was not accompanied by a parallel increase in apoptotic cells, as no significant differences in apoptosis were observed between HCT116 cells and the 14-fold resistant HCT-116 p21-/- cell line (figure  4 ). This suggests that modes of cell death other than apoptosis may operate in tumor cells following exposure to DOX, or, more generally, to DNA-damaging agents, a concept that is beginning to be proposed by a number of Authors [ 39 , 40 ]; of note, recent experimental evidence indicates p21 as one of the major determinants of terminal growth arrest induced by cytotoxic agents [ 41 - 43 ]. Therefore, although issues related to terminal growth arrest and senescence have not been specifically addressed in the present study, the possibility that these phenomena might play a role linking cell death to the observed increases in p21 levels should not be disregarded.The hypothesis that the cytotoxic response of the tumor cell lines tested in the present study may depend on p21 induction is further corroborated by data obtained following pre-treatment with the piperidine nitroxide TPL. The choice of this compound was dictated by previous findings indicating that TPL induces cell death in a number of tumor cell lines irrespective of their  p53  status [ 15 ], and that it increases p21 levels in  p53 -null cells [ 16 ]. The results of the present study show that TPL affects the four colon cell lines to similar extents, thus confirming that its growth inhibitory effect is independent of p53 function. HCT-116 p21-/- cells are actually slightly less responsive than the other cell lines [even though the difference does not attain statistical significance), which suggests the possibility that the effects of TPL are due in part to its ability to increase p21 levels. Interestingly, the nitroxide also induces p21 expression even in p53-deficient cell lines; this observation suggests that TPL can activate p53-independent pathways for p21 induction, as already noted following exposure of HT29 and HCT116 E6 cells to DOX. Moreover, activation of such pathways by TPL appears to sensitize tumor cells to the action of DOX: in fact, synergistic potentiation of DOX cytotoxicity is achieved by TPL in those cell lines where p21 expression can be induced, but only additive effects between TPL and DOX are observed in HCT116 p21-/-, where p21 expression is constitutively absent.p53p53Conclusions In summary, the results of the present study strongly suggest that 1) p21 induction may significantly contribute to the response of colon adenocarcinoma cells to DOX treatment; and 2) small molecules that can exploit p53-independent pathways for p21 induction, such as TPL, may find a place in chemotherapeutic protocols for the clinical management of colorectal cancer, where p53 function is often lost, due to genetic or epigenetic defects or to post-transcriptional inactivating mechanisms.
Conclusions
In summary, the results of the present study strongly suggest that 1) p21 induction may significantly contribute to the response of colon adenocarcinoma cells to DOX treatment; and 2) small molecules that can exploit p53-independent pathways for p21 induction, such as TPL, may find a place in chemotherapeutic protocols for the clinical management of colorectal cancer, where p53 function is often lost, due to genetic or epigenetic defects or to post-transcriptional inactivating mechanisms.Competing interests The author(s) declare that they have no competing interests
Competing interests
The author(s) declare that they have no competing interestsPre-publication history The pre-publication history for this paper can be accessed here:
Pre-publication history
The pre-publication history for this paper can be accessed here: