Suppression of cell proliferation and telomerase activity in 4-(hydroxyphenyl)retinamide-treated mammary tumors

Andrezj Bednarek, Anne Shilkaitis1, Albert Green1, Ronald Lubet2, Gary Kelloff2, Konstantin Christov1,3 and C. Marcelo Aldaz

Department of Carcinogenesis, University of Texas MD Anderson Cancer Center, Smithville, TX 78957,
1 Department of Surgical Oncology, University of Illinois at Chicago, 840 South Wood Street (M/C 820), Chicago, IL 60612 and
2 DCPC National Cancer Institute, Bethesda, MD 20852, USA


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The detection of telomerase activity has been proposed as a biomarker of breast cancer development and progression. In this study, we used cell proliferation and telomerase in MNU (N-methyl-N-nitrosourea)-induced mammary carcinomas as targets for assessing the response of tumor cells to 4-(hydroxyphenyl)retinamide (4-HPR), a known inhibitor of mammary carcinogenesis in animal models and premenopausal women. In mammary tumors of rats treated for 1, 2, 4 or 6 weeks with 4-HPR, we observed that telomerase activity decreased progressively with the extension of 4-HPR administration. A marked reduction in telomerase activity was already observed by 2 weeks after treatment and the lowest level was found at 6 weeks after initiation of 4-HPR treatment. The changes in telomerase activity were preceded and accompanied by a significant decrease in the percentage of proliferating cells as evaluated by 5-bromodeoxyuridine (BrdU)-labeling. However, when the values of telomerase activity in the individual tumors were compared with the percentage of proliferating cells, no significant correlation was found. These data suggest that the decreased telomerase activity in the animals treated with 4-HPR is not a simple consequence of the changes in cell proliferation, but a more complex phenomenon involving different cellular mechanisms and pathways. The time-dependent and consistent decrease of telomerase activity in the tumors treated with 4-HPR suggests that, in addition to the percentage of proliferating cells, telomerase activity could also be used as an endpoint in breast cancer chemotherapy studies.

Abbreviations: 4-HPR, 4-(hydroxyphenyl)retinamide; BrdU, 5-bromodeoxyuridine; ITAS, internal standard; MNU, N-methyl-N-nitrosourea; TERT, telomerase protein component; TRAP, telomere repeat amplification protocol.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Over the last few years, information has been accumulated on the role of telomerase in tumor development and progression (13). High telomerase activity was observed in practically all human breast cancers, and we and others detected significant activity already at the level of breast carcinoma in situ (4,5). Very recently, expression of the telomerase protein component (TERT) in normal breast epithelium was demonstrated by in situ hybridization (6). It was further shown that the levels and number of cells expressing telomerase increased during mammary carcinogenesis (6). Similarly, we recently demonstrated constitutive expression of telomerase in estrogen-regulated rat tissues, including the mammary epithelium, indicating that the telomerase-expressing cells belong to the stem cell compartment. Furthermore, we showed that telomerase does not associate with proliferation per se, but rather with proliferative potential (7). An increase in the levels of telomerase activity has also been described in rat mammary carcinomas (8).

Little is known about how various internal and external factors might affect telomerase activity in breast cells. It has been shown that telomerase activity is dramatically down-regulated in terminally differentiated cell populations (9,10). In vitro studies on human breast cancer cell lines showed that doxorubicin, cisplatinum and tomozolomide decreased telomerase activity, and this was dose- and time-dependent (11). Cells resistant to doxorubicin and tomozolomide showed no decline in telomerase activity and cell growth. Recently, in one clinical study, a limited number (n = 25) of patients with advanced breast carcinomas treated with chemotherapy showed a significant decrease in telomerase activity in their tumors when compared with untreated controls (12). These data suggest that the levels of telomerase activity in breast carcinomas could be used as a potential marker for assessing the effect of anti-neoplastic agents or of specific inhibitors of telomerase activity. Previous studies showed that 4-(hydroxyphenyl)retinamide (4-HPR) is a potent inhibitor of mammary carcinogenesis in animal models (13,14). It was also found that 4-HPR can induce regression and retardation of the growth of N-methyl-N-nitrosourea (MNU)-induced mammary carcinomas in rats (15). Very recently, Bischoff et al. (16) reported that targretin, a retinoid that is a retinoid X-receptor ligand, can also suppress the growth of established mammary carcinomas. These studies open a new avenue for potential implementation of retinoids not only for chemoprevention, but also for treatment of breast cancer. So far, no data have been published about whether 4-HPR or other retinoids can influence the levels of telomerase activity in mammary carcinomas, and whether this might correlate with changes in cell proliferation. Here, we report that 4-HPR dramatically suppresses cell proliferation and telomerase activity in MNU-induced mammary carcinomas, and that the longer the administration of 4-HPR, the lower the values of telomerase activity.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Animals
Female, virgin Sprague–Dawley [Hsp: (SD/BR)] rats were obtained from Harlan Sprague–Dawley (Indianapolis, IN) at 35 days of age, and after 1 week of quarantine were randomized by weight and injected i.p. twice with MNU, at the ages of 43 and 50 days. The animals were fed 4% Purina Chow AIN-76A diet (Teklad, Madison, WI) ad libitum and had free access to water. The weight of the animals was measured once a week.

Chemical carcinogen
MNU was obtained from Ash Stevens Inc. (Detroit, MI), dissolved in sterile acidified saline (pH 5.0) and injected i.p. at dose of 50 mg/kg body wt as indicated above. Two doses of MNU were used in order to increase the number of tumors.

4-(Hydroxyphenyl)retinamide
4-HPR was obtained from R.W.Johnson Pharmaceutical Research Institute (Spring House, PA) and was added to the diet at 782 mg/kg (2 mM) diet. Animals with palpable (4–8 mm) mammary tumors were randomized and treated for different time intervals (1, 2, 4 and 6 weeks) with 4-HPR. Placebo diet containing the 4-HPR vehicle only was given to the control animals.

Tumor volume
Tumors were measured twice weekly, and tumor volume (V, mm3) was calculated by the values of length (L), width (W) and thickness (T) of the tumors:

V = {pi}/6xL x W x T (mm3)

The animals were killed by CO2 narcosis, and tumors were removed and cut into two halves. One part of the tumor was fixed in 10% neutral formalin for histomorphology and immunocytochemistry, and the other part was frozen in liquid nitrogen for assessment of telomerase activity.

Cell proliferation
Proliferating cells in mammary carcinomas were labeled by 5-bromodeoxyuridine (BrdU, 50 mg/kg body wt; Sigma, St Louis, MO). The animals were killed 2 h after i.p. administration of BrdU. The nuclei labeled with BrdU were identified by an anti-BrdU monoclonal antibody (Beckton Dickinson, Palo Alto, CA) and ABC kit (Vector, Burlingame, CA), as described earlier (17). More than 1000 randomly distributed cells were scored from each tumor and the percentage of BrdU-labeled cells was expressed as BrdU-LI.

Telomerase activity assay
For telomerase detection, we used the PCR-mediated telomere repeat amplification protocol (TRAP) (18). As a positive control, an extract from cells with known telomerase activity (human breast cancer line MDA-MB-157, 100 cells equivalent) was used. As a negative control, cell extract was substituted for lysis buffer. As an additional control for the TRAP assay, we used an internal standard (ITAS; a gift of Dr J.Shay) that amplifies from the same primers (18). This internal standard, which consists of a 150 bp DNA product, allows identification of false-negative tumor samples that might contain Taq polymerase inhibitors. Cell extracts were obtained and TRAP was performed as previously described (18) with minor modifications (19). Two microliters of tissue extract (protein concentration 0.5 µg/µl) were used per assay. The CX primer, ITAS and Taq DNA polymerase (7 U per assay) were added to each sample after 5 min incubation at 90.8°C to make a hot start. Aliquots (10 µl) of the PCR mixture were analysed on 0.4 mm, 8% non-denaturing acrylamide gels (20x40 cm), run in 0.5xTBE buffer until the xylene cyanol had migrated 17 cm from the origin. The gels were then dried and exposed for 20 h to hyperfilm MP films (Amersham, Arlington Heights, IL). Following autoradiography, gels were also analysed after overnight exposure with a Molecular Dynamics PhosphorImager (Sunnyvale, CA). The area of integration of all peaks was normalized to the signal from the internal standard, then, after background subtraction, expressed relative to the positive control signal (100 cell equivalent) that was run with each experiment. The method described is only semi-quantitative, but it is sufficient for the comparative analysis of tumors relative to the same positive control cell extract.

Statistical analysis
The comparison of mean values between the different groups was evaluated by ANOVA with Fisher's LSD test.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
4-HPR suppresses the growth of established mammary carcinomas
The changes in tumor volume in control and 4-HPR-treated animals are presented in Figure 1Go. 4-HPR treatment was initiated when the tumors were small in size (4–8 mm). Two weeks of treatment with 4-HPR were not sufficient to suppress tumor growth. However, with extension of 4-HPR administration (4–6 weeks), the growth potential of tumors decreased compared with those in control animals. Among the animals treated with 4-HPR a substantial variability in tumor volume was observed with a significant decrease (P < 0.01) in the animals killed 6 weeks after initiation of 4-HPR administration only (Table IGo). The weight of the animals remained close for all time points with no statistical difference (P > 0.05) between control and 4-HPR treated groups (Table IGo), indicating that 4-HPR at 2 mM/kg diet is not toxic and does not affect growth.



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Fig. 1. Effect of 4-HPR on the growth of MNU-induced tumors. Note the decrease in tumor growth potential in the animals treated with 4-HPR as compared with the control non-treated animals. Ten tumors from the control group were followed for 6 weeks and the mean values of their volume evaluated. In 4-HPR treated groups animals were killed 1 (No. 9), 2 (No. 12), 4 (No. 5) and 6 (No. 7) weeks after initiation of treatment, and their weight (g) and the size of tumors (mm3) were measured (Table IGo).

 

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Table I. Effects of 4-HPR on body size weights
 
4-HPR inhibits cell proliferation in mammary carcinomas
BrdU-labeled cells in the control tumors were either randomly distributed among the tumor parenchyma or were located close to the stroma (proliferative zone, Figure 2aGo). Viable tumor cells lacking proliferating activity were frequently identified between the proliferating peripheral zones and the central necrosis. In the animals treated with 4-HPR, an overall reduction in proliferating cells was found (Figure 2bGo). However, myoepithelial and stroma cells continued to proliferate, indicating that 4-HPR inhibits mostly the epithelial cell proliferation. Among tumor parenchyma, there were areas lacking BrdU-labeled cells, as well as areas with a high number of labeled cells, suggesting that tumor cells are differentially sensitive to the antineoplastic effect of 4-HPR. In the control tumors, BrdU-LI was in the range between 15 and 38% with a mean value of 26.2 ± 6.2%, whereas in the animals treated with 4-HPR a significant decrease in BrdU-LI for all time points examined (1, 2, 4 and 6 weeks of treatment) was found. The differences in BrdU-LI values between various time points were insignificant (P > 0.05). In most of the animals treated with 4-HPR a significant variability in BrdU-LI values was observed. For instance, in the animals treated for 2 weeks with 4-HPR there were tumors with BrdU-LI below 1%, as well as above 15% (Figure 3aGo). In most tumors 4-HPR suppressed cell proliferation in the peripheral tumor areas where tissue disintegration was also observed (Figure 2bGo).



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Fig. 2. (a) A high number of BrdU-labeled cells in a control tumor with histology of adenocarcinoma. Most BrdU-labeled cells are located close to the basal membrane. The slide is counter-stained by haematoxylin, x200. (b) A decrease in the number of BrdU-labeled tumor cells in animals treated for 2 weeks with BrdU. The tumor is adenocarcinoma and has histology similar to those of control tumor (a). There are areas of tissue disintegration (arrows). The slide is counter-stained by haematoxylin, x200.

 


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Fig. 3. (a) Effect of 4-HPR treatment on BrdU-LI (%) in mammary tumors. Each point (empty circles) represents the BrdU-LI value for individual tumors. Mean values as indicated (filled circles); bars, SE. All 4-HPR treatment groups showed a statistical significant difference from the control group (P < 0.0002, ANOVA, Fischer's LSD). (b) Effect of 4-HPR treatment of telomerase activity in rat mammary tumors. Telomerase activity is plotted as percentage of activity relative to the positive control (100 cell equivalent for telomerase positive cell line). Each circle represents the telomerase value for individual tumors. Note the progressive decrease in telomerase activity with the time of 4-HPR administration. No significant difference was found in the values of BrdU-LI between the animals killed 1, 2, 4 and 6 weeks after initiation of treatment with 4-HPR.

 
4-HPR causes a progressive decline in telomerase activity in mammary tumors
Telomerase activity as evaluated by the TRAP assay was fairly variable in tumors from control untreated rats. A uniform suppression of telomerase activity was observed paralleling the extent of 4-HPR treatment. By 2 weeks of treatment, a reduction in telomerase activity was evident in most tumors analysed and the lowest level of telomerase activity was found at 6 weeks after initiation of 4-HPR treatment with little variability in telomerase levels between tumors (Figures 3b and 4GoGo). Although telomerase activity decreased concomitant with inhibition of cell proliferation, we did not observe a statistically significant direct correlation with the levels of telomerase activity and cell proliferation analysed by non-parametric Spearman rank correlation. BrdU-LI data (see below), when compared with telomerase activity, suggest that 4-HPR initially inhibits cell proliferation and later decreases telomerase activity, since, by 1 week of 4-HPR treatment, BrdU-LI was significantly lower than the control values, although telomerase levels in most of the tumors was still high.



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Fig. 4. Telomerase activity in rat mammary tumors as detected by the TRAP assay. Each lane represents individual tumors. P100, telomerase activity positive control (100 cell equivalent from telomerase positive tumor line); P(HI), same positive control after heat inactivation, buffer, lysis buffer negative control; ITAS, internal standard. Note the marked decrease in intensity of the telomerase ladder signal in the comparison of mammary tumors from the 4-week 4-HPR treatment group versus the higher activity observed in the 2-week treatment group.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In this study, we assessed the anti-tumor effect of 4-HPR on MNU-induced mammary carcinomas. Endpoint biomarkers were tumor volume, cell proliferation and telomerase activity. In most previous studies (except one, ref. 15), 4-HPR has been used as an inhibitor of mammary carcinogenesis (i.e. chemopreventive), but not of tumor growth (14). We observed that 4-HPR, when given in the diet of animals with established mammary carcinomas, suppresses cell proliferation and telomerase activity. To the best of our knowledge, this is the first study reporting the effect of 4-HPR on cell proliferation and telomerase activity in established mammary carcinomas in rats. In a recent study, which is still in progress, we observed that, in MNU-induced mammary carcinomas, between 20 and 35% of tumor cells were in the S-phase of the cell cycle, as labeled by BrdU, and that most of these cells were localized close to the stroma or basal lamina (20). We also found a very low number of apoptotic cells within the proliferating tumor areas and an increase in apoptotic cells in the areas close to necrosis (21). We also reported that 4-HPR selectively suppresses the hyperplastic and premalignant stages of mammary carcinogenesis (21). In this study, we observed that 4-HPR, within 1–2 weeks of treatment, significantly suppressed cell proliferation in mammary carcinomas. With extension of 4-HPR treatment for 4 and 6 weeks, the values of BrdU-LI appeared to increase (compared with the 2 week values) in some tumors, suggesting development of resistance of tumor cells to 4-HPR.

We also found that, in the animals treated with 4-HPR, telomerase activity decreased in a time-dependent fashion, with the lowest values 6 weeks after initiation of treatment, when the experiment was terminated. It appears that the decrease in telomerase activity is preceded and accompanied by decrease in cell proliferation. However, the increase in cell proliferation in some tumors in the animals killed 4 and 6 weeks after initiation of treatment with 4-HPR (when telomerase activity in the tumors remained low), and lack of strong correlation between the values of BrdU-LI and telomerase activity in individual tumors suggest that other factors, in addition to cell proliferation, are associated with telomerase activity regulation.

It has been reported that telomerase is variably expressed in MNU-induced mammary carcinomas (8). Here, we observed that the heterogeneity in telomerase activity was still very significant within 1 week of treatment, but was reduced after 6 weeks of 4-HPR administration, with all tumors showing low levels of telomerase activity (Figure 3bGo). These data suggest that 4-HPR causes a marked decline in the number of telomerase-expressing cells and, perhaps, in the levels of expression, bringing the enzyme activity to a baseline level that seems to be similar in all treated tumors. The half life of telomerase is apparently quite long (22) and this is important, considering that the decrease in activity we observed was slow and progressive, rather than abrupt, which would be expected if cell death is the major mechanism of eliminating telomerase-expressing cells (11). It is possible to speculate that 4-HPR predominantly eliminates tumor cell subpopulations exhibiting high proliferative potential and high levels of telomerase expression, and only cells with low residual activity remained after treatment. Using in vitro systems, other investigators have previously demonstrated a pronounced down-regulation of telomerase activity as a consequence of induction of differentiation by retinoic acid (9,10,23). It has also been shown that retinoids induce morphological and functional differentiation of normal rat mammary epithelial cells (24). Here, we demonstrate that retinoids in established mammary carcinomas also cause a marked decline in telomerase activity, which is preceded by a sharp decrease in cell proliferation. This allows speculation that, in addition to a possible selective killing of cells with high proliferative potential, the decrease in telomerase activity observed in tumors from 4-HPR treated animals may also be the consequence of induction of cellular differentiation and withdrawal from the pool of proliferating cells.

In conclusion, the results obtained in this study suggest that, in addition to cell proliferation, telomerase activity could also be used as a potential endpoint biomarker in assessing the effect of 4-HPR and probably other retinoids on experimental and possibly human breast cancer.


    Acknowledgments
 
This work was supported by a grant from the Cancer Research Foundation of America and Contract No. NO1-CN-55179-MAO from the NCI, Chemoprevention Branch (to K.C.). Part of these data were presented at the 1998 AACR Meeting in New Orleans


    Notes
 
3 To whom correspondence should be addressedEmail: christov{at}uic.edu Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received September 24, 1998; revised December 17, 1998; accepted December 30, 1998.