The effect of lithocholic acid on proliferation and apoptosis during the early stages of colon carcinogenesis: differential effect on apoptosis in the presence of a colon carcinogen

Vassiliki Kozoni1,2, George Tsioulias1, Steven Shiff1,2 and Basil Rigas1,2,3

1 Cornell University Medical College and
2 Rockefeller University, New York, NY 10021, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Lithocholic acid (LCA) is implicated in human and experimental animal carcinogenesis. Its effect on apoptosis and proliferation of the colonic epithelium was studied in a 1,2-dimethylhydrazine (DMH)-induced murine carcinogenesis model. Four groups of mice, control, LCA, DMH and DMH+LCA, were studied for 4 weeks, a period corresponding to early stages of carcinogenesis. Apoptosis (AI) and proliferation (PI) indices in the colon were determined by immunohistochemistry. LCA stimulated apoptosis [AI = 1.2 ± 0.3% (all values are the mean ± SEM) versus control 0.5 ± 0.1%, P < 0.05], as did DMH (4.3 ± 0.8%, P < 0.02). DMH increased apoptosis at the base of the crypt nearly 50-fold, with no effect at the lumenal third. In mice receiving DMH, LCA suppressed apoptosis almost completely (0.1 ± 0.03%); this suppression was complete at the lower two-thirds of the crypt (AI = 0) and 60% at the lumenal third. LCA increased proliferation (PI = 22.2 ± 4.6% versus 15.4 ± 1% in controls), but this did not reach statistical significance. DMH increased proliferation (PI = 34.6 ± 2.3%, P < 0.01). In mice receiving DMH, proliferation (41 ± 2.9%) was about two-thirds of the additive effect. LCA affected proliferation, mainly in the middle third of the crypt; DMH's effect was similar in distribution, but more pronounced. In mice receiving DMH, LCA shifts proliferation upward, extending it to the lumenal third of the crypt. LCA's main cell kinetic effect in the colon is on apoptosis; this effect differs in normal (stimulation) and pre-malignant colon (nearly complete suppression). LCA does not significantly stimulate proliferation in either normal or pre-malignant colon. The differential effect of LCA on apoptosis in the presence of a carcinogen partially explains its effect as a promoter on colon carcinogenesis in animal models, and may have important implications for human carcinogenesis.

Abbreviations: AI, apoptosis index; DMH, 1,2-dimethylhydrazine; LCA, lithocholic acid; PCNA, proliferating cell nuclear antigen; PI, proliferation index; RT, room temperature; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Numerous factors have been implicated in the etiology of colon cancer, the second most common fatal malignancy in the Western world. Among the most important of them are genetic abnormalities, a Western-style diet and bile acids (reviewed in ref. 1). Over the past three decades, many studies have demonstrated consistently that in populations at high risk of colon cancer the fecal bile acid concentration is elevated (reviewed in refs 2–4). A large body of evidence implicates bile acids such as cholic acid, lithocholic acid (LCA), deoxycholic acid and chenodeoxycholic acid as tumor promoters (512).

Effects of bile acids thought to influence colon carcinogenesis (reviewed in ref. 13) include loss of colonic surface epithelium (14), DNA damage (15), induction of cell proliferation (16), increased ornithine decarboxylase activity (17), suppression of the expression of HLA genes (1820), activation of protein kinase C (21), increased cell membrane permeability (22), control of gene transcription (gadd 153) (23) and inhibition of DNA polymerase ß (24). Another important effect of bile acids is the induction of apoptosis, which plays a critical role in colon carcinogenesis (15,2527).

Under normal conditions, cell mass homeostasis represents a tightly controlled balance between cell renewal and cell loss; neoplasia results from the dysregulation of either or both of these processes (28). In the colon, apoptosis normally takes place at the lumenal end of the crypts and proliferation at their base (29). In adenomatous colonic mucosa, this pattern is reversed: compared with normal, the lumenal surface displays twice the proliferative activity and the base half the apoptotic activity (30). The various stages of colonic carcinogenesis are related to increased rates of cell proliferation and progressively reduced rates of apoptosis (31,32).

The relationship between bile acids and apoptosis seems to be important for the development of colon cancer. Compared with colonic epithelial cells from normal individuals, normal-appearing colonic epithelial cells from patients with either colon cancer or adenoma are resistant to (ex vivo) bile-acid-induced apoptosis (26).

We sought to elucidate the effect of LCA on proliferation and apoptosis during the early stages of colonic carcinogenesis, i.e. before the apparent histopathological manifestations of adenomatous dysplasia and cancer. To achieve this, we evaluated the effect of LCA in BALB/c mice treated for 4 weeks with 1,2-dimethylhydrazine (DMH). DMH-treated mice provide an excellent animal model of colon cancer (3336).

We assessed not only the levels of proliferation and apoptosis in the colon, but also the topography of proliferating and apoptotic cells within the colonic crypts. The colonic crypt is an active unit of cells that proliferate and die in a sequence that maintains its architecture and ordered functions. This heterogeneity of the crypt made it necessary for us to assess the effects of LCA in its subcompartments.

This paper presents the results of our work, which shows that the effect of LCA on colonocyte kinetics in normal mice is different from that in mice undergoing carcinogenic changes in their colon.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Animals and treatment schedule
Twenty 10-week-old male Balb/c mice were used, divided into four groups. They were caged in groups of five, maintained in temperature- and humidity-controlled conditions in a 12 h dark and 12 h light regimen, and given food and water ad libitum.

The first group, which was the control group, was administered subcutaneously and transrectally the corresponding vehicles in a regimen identical to that of the other study groups. The second group was treated with 0.3 ml of a 2 mg/ml DMH solution, injected subcutaneously once a week and transrectal vehicle enema twice a day. The third group was treated with 0.5 ml of 5 mM LCA enema twice a day and injected subcutaneously with vehicle once a week. The combination of DMH and LCA at the above doses was administered to the fourth group of mice.

At the end of the fourth week of the experiment, all mice were killed and the distal 3 cm of the colon excised, opened along the longitudinal axis, thus exposing the mucosa, immediately immersed in cold Hank's Balanced Salt Solution (Sigma), irrigated thoroughly and stored in liquid nitrogen.

Determination of proliferation and apoptosis
Proliferation and apoptosis of colonic epithelial cells were determined histochemically, by staining for proliferating cell nuclear antigen (PCNA) and by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling (TUNEL) assay, respectively, as described (3739).

PCNA staining.
Fresh frozen colon specimens were thawed, fixed in 75% ethanol for 12 h and 95% ethanol for 24 h, embedded in paraffin and 4 µm paraffin sections were cut with a microtome so that the crypts were sectioned longitudinally from the base to the lumen. The sections were mounted on microscope slides, deparaffinized, rinsed with PBS, and incubated with 1% hydrogen peroxide for 30 min to block the endogenous peroxidase activity. They were then heated in a microwave oven for 1 min and incubated in 1% milk powder solution. The PC-10 clone PCNA antibody (Pharmingen, San Diego, CA) was applied at 1:200 dilution in 1% powdered milk for 1 h at room temperature (RT). The slides then received three 5 min washes with PBS, followed by incubation with the biotinylated secondary rabbit anti-mouse IgG (Pharmingen, San Diego, CA) for 30 min at RT, washing in PBS, and incubation with the tertiary avidin–biotin-complex reagent (Vector Laboratories, Burlingame, CA) for 30 min at RT. After rinsing with PBS, the slides were incubated with the chromogen 3,3'-diaminobenzidine (Sigma) at 1 mg/ml with 0.003% hydrogen peroxide for 3 min, then rinsed with distilled water, counterstained with hematoxylin, dehydrated and cover-slipped.

TUNEL staining.
TUNEL staining was performed in formalin-fixed, paraffin-embedded tissues that were cut 4 µm thick and deparaffinized. Endogenous peroxidase activity was quenched by hydrogen peroxide and nuclear protein was hydrolyzed by bathing the sections in proteinase K. Following this, sections were incubated with TdT buffer containing biotinylated d-UTP and TdT enzyme at 37°C for 1 h in a humid chamber; the reaction was terminated by washings with 2x SC (0.3 M NaCl, 0.003 M sodium citrate). Tissue specimens were incubated with serum albumin with additional normal horse serum and then extra avidin peroxide was added. After several washings, the slides were treated with DAB at room temperature and counterstained with hematoxylin.

Scoring.
We selected for scoring only crypts sectioned longitudinally through their entire length, with the base in contact with the muscularis mucosa and the upper part extending up to the lumen. Using a light microscope at x400 magnification, at least 30 crypts per animal were scored independently by two experienced investigators who were not aware into which group each specimen belonged. Cells with a blue nucleus were considered unlabeled, while those with a brown nucleus were considered labeled. To exclude the possibility that necrosis produced TUNEL-positive cells (instead of apoptosis), we carefully examined each field, applying the criteria established by Ben-Sasson et al. (40). In particular, (i) apoptosis is an ordered process, whereas `necrosis represents a chaos' (40) including all cellular elements of the mucosa at the affected site, and (ii) apoptosis is coupled with the elimination of dead cells by conventional routes; necrosis leads to the in situ accumulation of numerous dead corpuscles that display an unmistakable pattern of damage. In the tissue sections evaluated in this study, there was no evidence of necrosis, all TUNEL-positive cells being attributed to apoptosis.

Crypts were subdivided along their long axis into three equal-sized compartments: the upper (lumenal), the middle and the lower (basal). For this, the cells in each hemicrypt were counted and this number was divided by three to define the three equal compartments.

We calculated the proliferation index (PI) and apoptotic index (AI) for both the entire crypt and for each of its three compartments. This was obtained by dividing the number of labeled cells by the number of cells either of the entire crypt or of each of its compartments and multiplying by 100. Data from at least 30 crypts were used to calculate each index. In addition, the number of positive cells in each of the three zones along the longitudinal axis of the crypt was determined.

Statistical analysis
Data were analyzed by the Mann–Whitney non-parametric U test; P < 0.05 was considered statistically significant.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Histological findings
LCA caused loss of orientation of cells within the colonic crypts, mild nuclear atypia with disturbed cytoplasmic/nuclear ratio and occasional hyperchromasia of the nucleus with conspicuous nucleoli. The most obvious architectural change was the increased length of the crypts compared with controls (26.38 ± 1.32 versus 20.56 ± 1.59 cells, P < 0.03). Similar changes were noted in mice treated with DMH (25.65 ± 1.56). In animals treated with DMH, LCA caused more prominent loss of cellular orientation and also branching of crypts. In addition, the increased length of the crypts was maximal in this group as well (28.95 ± 0.82, P < 0.002, compared with control, but not different from the `LCA only' or `DMH only' groups).

Effect of LCA on apoptosis
Treatment of mice with LCA had a significant effect on apoptosis of the colonic epithelial cells (Table IGo; Figures 1, 3 and 4GoGoGo).


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Table I. The apoptotic index (AI) of colonocytes in mice treated with DMH or LCA or both and their control group
 


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Fig. 1. The effect of LCA on apoptosis in the colonic crypt of mice. The AI was determined as described in Materials and methods for both the entire crypt and its three equal-sized compartments: basal, middle and lumenal.

 


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Fig. 3. The effect of LCA on proliferation and apoptosis in the colon of mice. Proliferation (A–D) was determined by histochemical detection of PCNA antigen expression as in Materials and methods. (A) Control: proliferation predominates at the base of the crypt. (B) DMH treated: the proliferation zone is shifted upwards. (C) LCA treated: less prominent expansion of the proliferation zone. (D) LCA and DMH treated: the proliferation zone is further expanded upwards. Apoptosis (E–H) was determined by the TUNEL method, as in Materials and methods. (E) Control: apoptosis is restricted to the lumenal compartment. (F) DMH treated: apoptotic cells are evident in the lower two-thirds. (G) LCA treated: prominent apoptosis in the lumenal third. (H) LCA and DMH treated: apoptosis is severely suppressed, with only a single apoptotic cell in this field. x100.

 


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Fig. 4. The effect of LCA on apoptosis and proliferation in the colonic crypt of mice. Schematic diagram of a colonic crypt and its subdivision into three compartments. The tables present the AI and PI per crypt compartment for each group of animals. The relative contribution of apoptosis or proliferation in each crypt compartment is presented as a percentage of the apoptotic or proliferative activity, respectively, of the entire crypt.

 
In control animals, the AI of the entire crypt was 0.5 ± 0.1% (the mean ± SEM is given for this and all subsequent values). The AI varied substantially among the three compartments of the crypt. Thus, the lower third (base of the crypt) showed an AI of 0.3 ± 0.3%, the middle one 0%, while that of the upper third (lumenal) was 1.4 ± 0.4%. These differences are statistically significant and quantitatively important.

Treatment with LCA increased the AI of the entire crypt to just a little over twice normal compared with control (1.2 ± 0.3% versus 0.5 ± 0.1%, P < 0.05). The distribution of apoptosis in the three compartments of the colonic crypt was uneven, with the AI being maximal in the lumenal compartment and minimal in the basal crypt compartment. LCA suppressed apoptosis at the base of the crypt compared with controls (0.1 ± 0.8 versus 0.3 ± 0.3). In the middle and lumenal thirds of the crypt, LCA increased the AI above controls (0.3 ± 0.2% versus 0% and 3.2 ± 0.9% versus 1.4 ± 0.4%, respectively). None of these changes were statistically significant.

Animals treated with DMH displayed changes in AI compared with controls. The AI of the entire crypt increased nearly 9-fold compared with control (4.3 ± 0.8% versus 0.5 ± 0.1%, P < 0.02). Of greater interest, this increase was not uniformly displayed along the length of the crypt. The strongest effect was at the base of the crypt, the AI being almost 50-fold greater than the corresponding control value (P < 0.01). In the middle third, there was a marginal increase in AI (0.3 ± 0.2% versus 0%). In the lumenal third of the crypt, apoptosis was totally suppressed, being undetectable (AI = 0). Compared with DMH, LCA had a much milder effect on apoptosis.

When given to animals treated with DMH, LCA had a profound effect on apoptosis in the colon. The AI of the entire crypt was suppressed compared with control (0.1 ± 0.03% versus 0.5 ± 0.1%, P < 0.03). This suppression was all the more pronounced when compared with either DMH or LCA groups (0.1 ± 0.03% versus 4.3 ± 0.8%, P < 0.01, and 1.2 ± 0.3%, P < 0.006, respectively). The greatest suppressive effect of LCA in the DMH-treated mice was manifest in the lower two-thirds of the crypt where apoptosis was totally eliminated; in the upper third, apoptosis was severely reduced (one-third of the normal value, P < 0.03).

Figure 4Go gives a schematic overview of both the absolute and relative effect of these compounds on apoptosis in each segment of the colonic crypt.

Effect of LCA on proliferation
In addition to the effect of LCA on apoptosis, we examined its effect on the proliferation of the colonic epithelial cells (Table IIGo; Figures 2–4GoGoGo).


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Table II. The PI of colonocytes in mice treated with DMH or LCA or both and their control group
 


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Fig. 2. The effect of LCA on proliferation in the colonic crypt of mice. The PI was determined as described in Materials and methods, for both the entire crypt and its three equal-sized compartments: basal, middle and lumenal.

 
In control animals, the PI of the entire crypt was 15.4±1% with no proliferative cells in the lumenal third of the crypt; all the proliferative cells were encountered in the lower two-thirds. The PI was 42.6 ± 2.8% at the base of the crypt where stem cells are located, and 1.9 ± 0.5% in the middle third.

LCA increased the PI of the entire crypt only marginally compared with control (22.2% versus 15.4%, difference not significant). Compared with control, the most pronounced increase occurred in the middle third of the crypt (13.2 ± 6% versus 1.9 ± 0.5%), but this change did not reach statistical significance either.

DMH increased proliferation compared with control animals. Thus, the PI of the entire crypt was over twice normal (34.6 ± 2.3% versus 15.4 ± 1%, P < 0.01). The proliferation zone extended from the base up to the lumenal third of the crypt. The increase in PI was more profound in the upper two-thirds when compared with control (2.6 ± 0.9% versus 0%, P < 0.01, in the lumenal compartment, and 32.8 ± 2.9% versus 1.9 ± 0.5%, P < 0.01, in the middle compartment). Changes at the base of the crypt were less pronounced (68.1 ± 0.7% versus 42.6 ± 2.8%, P < 0.01). Compared with the LCA group, the entire crypt of DMH-treated mice showed increased PI (34.6 ± 2.3% versus 22.2 ± 4.6%, P < 0.02). In the upper two-thirds of the crypt, the DMH group showed enhanced proliferation compared with the LCA group. This difference was more pronounced in the middle third of the crypt (32.8 ± 2.9% versus 13.2 ± 6%, P < 0.02).

The effect of LCA on proliferation became statistically significant only in mice treated with the carcinogen DMH. The PI of the entire colonic crypt of these mice was significantly increased compared with controls (41 ± 2.9 versus 15.4 +1, P < 0.006) or a 2.6-fold increase, and also compared with the LCA-only group of mice (41 ± 2.9% versus 22.2 ± 4.6%, P < 0.03), an almost 2-fold increase.

Compared with controls, the greatest increase was manifest in the upper two-thirds of the crypt (3.4 ± 0.7% versus 0%, P < 0.003, in the lumenal compartment, and 46.8 ± 5.7% versus 19.5 ± 0.5%, P < 0.006, in the middle compartment). Compared with the `LCA-only' group of animals, the PI was also increased, being 17-fold greater than the corresponding LCA value in the lumenal compartment (3.4 ± 0.7% versus 0.2 ± 0.2%, P < 0.01); less dramatic increases were noted in the other two crypt compartments.

Figure 4Go gives a schematic overview of both the absolute and relative effect of these compounds on proliferation in each segment of the colonic crypt.

The PI/AI ratio
The PI/AI ratio gauges the relative contribution of the two antithetic processes on cell homeostasis in the colonic crypt. Table IIIGo summarizes our calculations of the PI/AI ratio in the various groups of mice. Normally, proliferation predominates heavily over apoptosis in the basal two-thirds of the crypt; apoptosis predominates at the lumenal third. LCA shifts the predominance of proliferation to the lower part of the crypt, making it more pronounced at its basal third. Under DMH treatment, proliferation predominates over apoptosis throughout the crypt, such an effect being maximal in its middle third. When LCA is given to mice also receiving DMH, proliferation predominates throughout the crypt. Indeed, it appears that under such treatment the sole cell kinetic activity of the crypt is proliferative.


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Table III. The PI/AI ratio of mice treated with DMH or LCA or both and their control group
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Our findings provide an insight into how LCA may contribute to colon carcinogenesis via its effects on colonocyte kinetics. Our observations in a murine model of colon cancer are focused on the very early stages of malignant transformation (initiation and promotion), long before overt neoplastic changes become apparent.

In this model, the first histological changes, focal hyperplastic changes and focal atypias, appear on day 38 following initiation of treatment with DMH. Cancers, developing in practically all animals, first appear on day 135 (33). In our study, animals were killed 28 days after the start of DMH injection. Thus, the effect of LCA that we have observed is associated with the very early stages of the carcinogenic process, developing under the influence of an exogenous carcinogen.

Another feature of this study is that we assessed not only the levels of proliferation and apoptosis in the colon, but also the topography of proliferating and apoptotic cells within the colonic crypt. Each crypt is an active unit of cells that proliferate and die in a certain sequence that maintains its architecture and highly ordered functions. Ordinarily, colonocytes proliferate predominantly at the base of the crypt. As cells move towards the upper segments, they differentiate, lose the capacity to divide, and within several days die and are shed off. When an adenoma forms, the proliferating compartment expands all the way up to the surface. Dividing cells accumulate at the surface, new glands form, and the accumulation of neoplastic glands produces the polyp (41). Assessment of the effects of LCA on crypt subcompartments was necessary precisely because of such heterogeneity of the crypt.

When the colonic crypt is considered in its entirety, LCA stimulates apoptosis. A similar effect is observed in mice receiving DMH. However, when LCA is administered to mice also treated with the carcinogen DMH, apoptosis is profoundly inhibited to almost total suppression. These suppressed levels of apoptosis represent 20% of the normal value and 2% of the expected degree of apoptosis if the individual effects of each compound were additive.

When the subcompartments of the crypt were examined, the effect of LCA on apoptosis differs substantially among crypt compartments. Normally, four-fifths of apoptosis takes place at the upper third of the crypt, none at its middle and only one-fifth at its lowermost third. LCA administered to normal mice has a quantitatively modest effect on apoptosis. There is, however, a clear suppression of apoptosis at the base of the crypt. DMH alone increased apoptosis almost 9-fold in absolute terms, but virtually all of it occurred at the base of the crypt, with none noted at the lumenal third. This could be conceptualized as a protective response of cells exposed to a mutagen. When LCA was administered to mice also receiving DMH, no apoptosis at all occurred in the lower two-thirds of the crypt, and only minimal apoptosis was detected at the lumenal side

LCA alone has only a marginal effect on proliferation, which does not reach statistical significance. DMH alone, on the other hand, stimulates proliferation. When LCA is administered to mice who also are treated with the carcinogen DMH, its effect on proliferation is practically additive with that of DMH, being ~80% of the sum of their individual effects.

As expected, normally almost all of proliferation occurs at the base of the crypt. Animals treated with LCA show a predominance of proliferation at the base of the crypt, although there is a clear (6-fold) increase of proliferation in its middle third compared with controls. DMH shifts the proliferation upward, with about one-third of it now taking place in the middle crypt compartment, and ~3% in the lumenal third, a distribution more or less similar to that seen with LCA. When LCA is given to animals receiving DMH, the same upward shift of proliferative activity is maintained.

The PI/AI ratio that we calculated gauges the relative contribution of the two antithetic processes on cell homeostasis in the colonic crypt under the various treatments of the mice. The distribution of these processes in the crypt was the expected one, i.e. predominance of proliferation in the lower part of the crypt and of apoptosis in its lumenal part. LCA tended to accentuate this distribution, making the predominance of proliferation in the lower part of the crypt even greater. When LCA was administered to mice also receiving DMH, the most impressive change was the >100-fold change in the PI/AI ratio: instead of apoptosis, proliferation was predominating in the lumenal third of the crypt. Indeed, it appears that under such treatment the sole cell kinetic activity of the crypt was proliferative.

Overall, these data demonstrate that, in this model system, the presence of a carcinogen radically alters the effect of LCA on colonocyte kinetics. During the early stages of carcinogenesis, its modest pro-apoptotic effect is reversed, reaching an almost total suppression of apoptosis. However, there is no analogous effect on proliferation. The modest and statistically not significant proliferative effect of LCA is not altered in the presence of the carcinogen. In fact, LCA increased the PI of normal colon by 7 percentage points, precisely the increase it brought about in the colon of mice treated with DMH (34% to 41%).

The reasons for this differential activity of LCA are not immediately apparent. If we could define the crypts of animals treated with DMH as `pre-neoplastic', then one might speculate that the signaling pathway of apoptosis in these cells is altered in a way that it is no longer susceptible to the normally pro-apoptotic effect of LCA. Alternatively, one might consider that LCA is handled differently by these `pre-neoplastic' cells, thus losing its ability to affect the apoptotic pathway.

Whatever the mechanism of this change in the effect of LCA, these results provide an insight into how this compound may contribute to colon carcinogenesis. Evidence from rodent models of colon cancer indicates that LCA by itself does not cause colon cancer, even after prolonged administration of high concentrations. However, in the presence of DMH, LCA enhances tumor formation, roughly doubling the number of tumors formed in the presence of DMH alone (5,6). Our findings lend mechanistic support to the biological behavior of LCA. Alone, LCA increases the rate of apoptosis in the colon without a significant effect on proliferation. Thus, the enhanced apoptosis ensures that the colon responds appropriately to a damaging agent by eliminating cells that ought not to survive and proliferate. In contrast, in the presence of the carcinogen, LCA, by practically eliminating the ability of colonocytes to undergo apoptosis, facilitates the development of cancer.


    Notes
 
3 To whom correspondence should be addressed at: Rockefeller University, Box 330, 1230 York Avenue, New York, NY 10021, USA Email: rigasb{at}aol.com Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received June 29, 1999; revised December 23, 1999; accepted December 30, 1999.