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Cell Growth & Differentiation Vol. 11, 437-445, August 2000
© 2000 American Association for Cancer Research

Bcl-2 and Bax Are Differentially Expressed in Hyperplastic, Premalignant, and Malignant Lesions of Mammary Carcinogenesis1

Anne Shilkaitis, Jewell Graves, Rajeshwari R. Mehta, Lan Hu, Ming You, Ronald Lubet, Vernon Steele, Gary Kelloff and Konstantin Christov2

Department of Surgical Oncology, University of Illinois at Chicago, Chicago, Illinois 60612 [A. S., J. G., R. R. M., K. C.]; Department of Pathology, Medical College of Ohio, Toledo, Ohio 43699 [L. H., M. Y.]; and National Cancer Institute Division for Cancer Prevention, Bethesda, Maryland 20852 [R. L., V. S., G. K.]


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Previously, we found that vorozole (Vz), a nonsteroidal aromatase inhibitor, suppresses the development and progression of mammary tumors in rats. Here we evaluated for the first time the expression of cell death-related proteins Bcl-2 and Bax in hyperplastic, premalignant (carcinoma in situ), or malignant (carcinoma) lesions of mammary carcinogenesis; we also assessed whether these proteins are involved in mediating Vz-induced cell death in tumors. We found that Bcl-2 and Bax were equally expressed in epithelial cells of terminal end buds, ducts, and alveoli. However, in myoepithelial cells, the level of Bax expression was much higher than the level of Bcl-2 expression. Bcl-2 and Bax levels in hyperplastic lesions were similar to those of normal mammary epithelial cells but lower in most carcinomas in situ and carcinomas. In animals with established mammary tumors, Vz induced apoptotic cell death, which was primarily associated with a decrease in Bcl-2 and, to a lesser extent, with a decrease in Bax. These data support the hypothesis that Bcl-2 loss is more potent than Bax gain in regulating apoptotic cell death in mammary tumors.


    Introduction
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Over the last several years, a tremendous amount of information has been generated indicating that physiological ACD3 is an active, well-programmed cellular event involving activation and/or suppression of various genes and gene families (12) . Among these families, Bcl-2 and members of its family appear to protect the cells from death, whereas Bax and related proteins potentate cell death (34) . However, Bcl-2 and Bax are differentially expressed in various tissues and cell populations and may respond differentially to apoptotic stimuli (56) . In the human breast, Bcl-2 and Bax are constitutively expressed in epithelial cells but are variably expressed in HLs, CIS lesions, and carcinomas (78) . A positive correlation has been found between Bcl-2 expression and ER+ status of tumors, but no clear association has been shown between the levels of Bcl-2 expression, the metastatic potential of tumor cells, and clinical outcome (910) . In contrast, Bax-{alpha}, a splice variant of Bax that promotes apoptosis, is expressed in normal ductal and alveolar cells as well as in normal breast cell lines, but only weak expression or no expression of Bax-{alpha} has been detected in cancer cell lines and breast carcinomas (1112) . Another study using ICH reported that Bax is expressed in about 30% of breast carcinomas and that reduced Bax expression is associated with poor response of tumors to chemotherapy (13) . These data suggest that a deregulation in Bcl-2 and Bax synthesis and function occurs in breast cancer development and progression and that the level of expression of both proteins may affect the response of tumor cells to antitumor agents.

ACD in the mammary gland of rodents has been studied predominantly after ovariectomy or in weaning animals after removal of pups (1415) . Early (1–3 days) and late (4–10 days) phases of tissue remodeling have been characterized, and differential expression of certain genes/proteins within these phases has been identified (16-18) . Most alterations have been identified in genes/proteins involved in the regulation of ACD and in the functioning of intercellular matrix proteins (16171920) . Among the various genes, p53 appears not to be involved in mediating hormone-dependent cell death in the mammary gland of rodents; however, it might affect radiation-induced cell death, which is much higher in MECs of p53 knockout mice than in heterozygous mice (21) .

Very recently, data were published showing that gain of Bcl-2 is more potent than loss of Bax in regulating MEC survival in mice (22) . However, whether the same is valid for tumor cells is unknown. Mice with knockout stromolysin-1 gene showed increased ACD during the initiation phase of 7,12-dimethylbenz(a)anthracene-induced mammary carcinogenesis, and fewer tumors developed in mice with the knockout stromolysin-1 gene than in the control heterozygous mice (23) . Studies by Srivastava et al. (24) indicated increases in Bcl-x/s, TRPM-2, c-myc, and p53 in 7,12-dimethylbenz(a)anthracene-induced mammary tumors treated with chorionic gonadotropin, suggesting that tumor regression is probably associated with alterations in the expression of the above-mentioned genes and proteins. We also observed that Vz-induced tumor regression is associated with increased ACD and non-ACD and decreased cell proliferation (25) . Studies, mostly on breast tumor cell lines, have shown that most cytostatics kill tumor cells by ACD and that this has been associated with modulation of the expression of members of the Bcl-2 and/or Bax families (26-28) . However, it is difficult for this to be proven in human tumors because serial biopsies need to be taken from tumors in the course of treatment. Most solid tumors are also composed of heterogeneous cell populations, which may respond differentially to cytostatics or may also develop resistance (2829) . To overcome some of these difficulties, we initiated this study to assess the feasibility of MNU-induced mammary tumors in rats as a model system for evaluating the relationship between the response of tumor cells to chemopreventive and antitumor agents in vivo and the expression of genes/proteins involved in the regulation of cell death (25) . Previous studies have shown that the MNU-induced mammary carcinogenesis model used in this study is relevant in many aspects to the development of human breast cancer (3031) .

In the present study, we tested the hypotheses that malignant transformation of MECs in rats is associated with deregulation of Bcl-2 and Bax expression and that both proteins are differentially involved in modulation of Vz-induced ACD in mammary tumors. We found for the first time that, compared with normal ductal cells, expression of both Bcl-2 and Bax decreased in the late stages (CIS and carcinoma) of mammary carcinogenesis and that Vz-induced cell death is primarily associated with a decrease in Bcl-2 and only secondarily associated with a decrease in Bax. Surprisingly, we found that both Bcl-2 and Bax are overexpressed in apoptotic cells, suggesting their differential involvement in the early and late stages of ACD.


    Results
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
In various normal mammary structures and cell populations as well as in HLs and CIS, Bcl-2 and Bax expression was evaluated by ICH only. In tumors, Western and Northern blot analyses were used in addition to ICH. Four groups of animals were examined: (a) control young (35- and 50-day-old) and adult (110-day-old) rats; (b) rats sacrificed 4–6 weeks after MNU administration, when hyperplastic and premalignant (CIS) lesions occurred in the mammary gland; (c) rats sacrificed 9 weeks after carcinogen administration, when, in addition to HLs and CIS, tumors developed; and (d) animals with established mammary tumors treated with Vz (Table 1Citation ).


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Table 1 Bcl-2 and Bax expression in normal mammary gland structures, hyperplastic lesions, CIS, and tumorsa

 
Bcl-2 and Bax Are Differentially Expressed in Normal Mammary Gland Structures and Cell Populations.
Mammary glands of young rats and adult rats differed in architecture. In young mature (35- and 50-day-old) rats, TEBs and ducts predominated, and few alveoli were observed (Fig. 1Citation A, short arrow). These morphological structures are composed of epithelial and myoepithelial cells surrounded by stroma cells (fibroblasts, adipocytes, and endothelial cells). In TEBs, in addition to epithelial (body) and myoepithelial cells, cap cells were also identified. Bcl-2 was expressed in the cytoplasm of body and cap cells (scores, 2+ and 3+), but the level of expression was lower in myoepithelial cells (scores, 1+ and 2+; Fig. 1ACitation ). Bcl-2 was also identified in fibroblasts, adipocytes, and endothelial cells (scores, 1+ and 2+). In contrast to Bcl-2, Bax was highly expressed in the cytoplasm of myoepithelial and cap cells (Fig. 1BCitation , arrow; score, 4+) and showed medium or strong expression in body cells, fibroblasts, and endothelial cells (scores, 2+ and 3+; Fig. 1BCitation ). No difference was found in the levels of Bcl-2 and Bax expression in young (35- and 50-day-old) and adult (110-day-old) animals. ACD, as measured by the AI, was low in both epithelial and myoepithelial cells (AI = 0.3 ± 0.1% versus 0.2 ± 0.1%, respectively), without significant differences between both cell types (P < 0.1), indicating that the differences in Bcl-2 and Bax expression are not related to differences in AI.



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Fig. 1. A, Bcl-2 expression as evaluated by ICH in normal mammary gland of a 50-day-old rat. Bcl-2 is almost equally expressed in TEBs (arrow), in a branching TEB (small arrow), and in a longitudinally sectioned duct (left corner). Note that there is no difference in Bcl-2 expression between epithelial and myoepithelial cells. The slide is counterstained with hematoxylin (x100). B, in a parallel section, Bax expression is presented in the same morphological structures as in A. Bax is expressed much more prominently in myoepithelial cells than in epithelial cells of TEBs (arrow) and of the other normal mammary structures. The slide is counterstained with hematoxylin (x100). C, Bcl-2 expression in mammary hyperplastic (h) and premalignant (arrow) lesions. The animal was sacrificed 6 weeks after carcinogen (MNU) administration. Bcl-2 expression in the above-mentioned lesions (score of 3+ according to the semiquantitative scale; see "Materials and Methods") appears lower than that in normal ductal structures (small arrow). The slide is counterstained with hematoxylin (x100). D, Bax expression in similar mammary premalignant lesions (CIS) as in C. This is the same animal as in C. The lesions were classified as ductal CIS and in morphology appear similar to the noncomedocarcinoma type of ductal CIS in the human breast. The level of Bcl-2 expression in the lesions (score, 3+) appears lower than that in normal ducts (arrow; score, 4+). The slide is counterstained with hematoxylin (x100). E, strong Bcl-2 expression (score, 3+) in the periphery of an adenocarcinoma. The staining decreased in the central tumor areas. The animal was sacrificed 10 weeks after carcinogen administration. Bcl-2 expression in normal alveolar cells (arrow) is higher (score, 4+) than that in tumor cells (score, 2+). The slide is counterstained with hematoxylin (x100). F, Bax expression in a parallel section from the same tumor as in E. The level of Bax expression in the tumor periphery is again higher than that in the central tumor areas. There is no central necrosis. The level of Bax expression in normal alveolar cells (arrow) is higher than that in tumor cells. Bax expression in myoepithelial cells (arrow) is higher than that in epithelial cells as well as that in tumor cells. The slide is counterstained with hematoxylin (x100). G, Bax expression in an adenocarcinoma in an animal treated for 4 days with 2.5 mg Vz/kg b.w. Mammary tumor was induced by MNU, and when the tumor reached 10 mm in diameter, Vz was given by gavage. Note the prominent staining of myoepithelial cells (arrow) and the relatively strong staining of tumor cells (score, 3+). The slide is counterstained with hematoxylin (x100). H, a double staining of a mammary carcinoma for apoptosis and Bcl-2 expression. The animal was treated for 10 days with 2.5 mg Vz/kg b.w. Apoptotic cells are stained brown by the TUNEL assay (see "Materials and Methods"). The red staining is for Bcl-2 protein (AEC staining). Note the low level of Bcl-2 in apparently viable tumor cells and the strong staining in apoptotic cells (arrow). The slide is counterstained with hematoxylin (x400). I, a double staining of a Vz-treated tumor (10 days of treatment) for apoptosis and Bax expression. Bax is expressed (red staining) in both peripheral and central tumor areas (small arrow). Note the brown-stained nucleus of apoptotic cells (TUNEL assay) and the red-stained cytoplasm for Bax (short arrow). The slide is counterstained with AEC. Hematoxylin x400.

 
Bcl-2 and Bax Are Differentially Expressed in HLs, CIS, and Carcinomas.
One objective of this study was to identify potential differences in Bcl-2 and Bax expression in hyperplastic and premalignant (CIS) stages of mammary carcinogenesis. HLs and CIS preceded the occurrence of mammary tumors and were identified in mammary gland of 20 animals sacrificed 4–6 weeks after MNU administration (Fig. 1C and D)Citation . These lesions were also found among the mammary parenchyma of animals with established mammary tumors. HLs were divided into three categories: (a) TEBH, in which the hyperplastic process developed exclusively in TEBs; (b) DH, in which HLs occurred in ducts and ductal branching areas; and (c) AH, in which the hyperplastic process occupied the alveolar structures. In morphology, TEBH and DH were similar to nonatypical DH, whereas AH was close to lobular hyperplasia of the human breast. Of 42 HLs examined, 22 were TEBHs, 15 were DHs, and 5 were AHs (Table 1)Citation .

In evaluating the levels of Bcl-2 and Bax expression in various mammary lesions and tumors, we used as reference values those of epithelial cells in the normal ducts and alveoli surrounding the lesions. Most HLs had medium (2+) or high (3+) levels of Bcl-2 and Bax expression (Fig. 1CCitation ), whereas CIS (Fig. 1DCitation ) and carcinomas had low (1+), medium (2+), or high (3+) levels of Bcl-2 and Bax expression (Table 1)Citation . Myoepithelial cells surrounding various lesions and tumors had very high (4+) Bax expression and low (1+) or medium (2+) Bcl-2 expression (Fig. 1Citation C, short arrow).

The levels of Bcl-2 or Bax expression in epithelial cells of various HLs (TEBH, DH, and AH) were similar or close, suggesting that the place of origin and histomorphology of HLs do not affect the expression of both proteins. Summarized data of Bcl-2 and Bax expression in various mammary gland lesions and tumors are given in Table 2Citation . No significant difference in Bcl-2 and Bax expression was found between normal MECs and HLs (Table 2)Citation : Bcl-2 was 3.1 ± 0.5 in normal MECs versus 2.9 ± 0.4 in HLs (P > 0.1), and Bax was 3.2 ± 0.6 in MECs versus 2.9 ± 0.7 in HLs (P > 0.1), respectively. However, in CIS, a significant decrease in both Bcl-2 (P < 0.01) and Bax (P < 0.05) was found. The levels of Bcl-2 and Bax expression in carcinomas were lower than those in normal MECs or HLs (P < 0.01 and P < 0.001, respectively). No significant difference was found in both Bcl-2 expression and Bax expression between CIS and carcinoma (P > 0.1 and P > 0.1, respectively).


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Table 2 Bcl-2 and Bax expression in HLs (TEBH, DH, and AH), CIS, and carcinomas (CA)a

 
Histology of Mammary Tumors Does Not Affect Bcl-2 and Bax Expression.
We also compared the levels of Bcl-2 and Bax expression between various histological types of mammary carcinomas. Forty-four tumors were divided into three groups: (a) adenocarcinomas; (b) papillary carcinomas; and (c) comedocarcinomas (Table 3Citation ). The most frequent were adenocarcinomas and papillary carcinomas. In some tumors, mixed structures (adenocarcinoma + papillary or adenocarcinoma + comedocarcinoma) were observed. In these tumors, the prevailing structure was considered for classification. In control tumors, Bcl-2 and Bax were variably expressed among tumor parenchyma (Fig. 1E and F)Citation . In most tumors, independent of their histology, scores of 2+ and 3+ predominated. Few tumors had scores of 1+ or 4+ (Table 3)Citation . When the average values of Bcl-2 or Bax expression were compared, no significant difference between various histological types was found.


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Table 3 Histomorphology of tumors and level of Bcl-2 and Bax expressiona

 
Contrary to normal mammary structures and HLs, in which Bcl-2 and Bax were almost evenly expressed in epithelial cells, in CIS and particularly in tumors, the expression of both proteins among the tumor parenchyma varied (Fig. 1Citation , C-F). Bcl-2 and Bax were expressed in both peripheral and central tumor areas; however, the level of Bcl-2 expression was lower in the areas close to necrosis, whereas Bax expression remained the same or even increased. Bax was also highly expressed in myoepithelial cells surrounding the lesions and tumors (Fig. 1Citation G, arrow).

The variability in Bcl-2 and Bax expression in tumors was also confirmed by Western blot analysis (Fig. 2Citation ). Eight nonselected control tumors (the values of four are presented in Fig. 2ACitation , Lanes 1–4) and eight Vz-treated tumors (the values of four are presented in Fig. 2ACitation , Lanes 5–8) were examined by ICH and Western blot, and the values for Bcl-2 and Bax expression were compared. In control tumors, the values of Bcl-2 were more variable than the values of Bax. This variability further increased in Vz-treated animals (Fig. 2ACitation , Lanes 5–8).



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Fig. 2. Effects of Vz on Bcl-2 and Bax expression in mammary tumors. A, Western blot analysis of Bcl-2 and Bax in control and Vz-treated tumors. Jurkat cells were used as a positive control for Bcl-2 (29 K) and Bax (23 K) expression. Lanes 1–4 are samples from control tumors, Lanes 5 and 6 are tumors treated for 4 days with 2.5 mg Vz/kg b.w., and Lanes 7 and 8 are tumors treated for 10 days with 2.5 mg Vz/kg b.w. Both proteins are variably expressed in control and Vz-treated tumors. The level of Bcl-2 expression in Vz-treated tumors (Lanes 5–8) appears lower than that in control tumors. Bax is relatively equally expressed in control tumors. The level of Bax expression even increased in some Vz-treated tumors (Lanes 5 and 6) or remained the same as in the control tumors (Lanes 7 and 8). B, Northern blot analysis of Bcl-2 and Bax in mammary tumors. The mRNA expression of Bcl-2 and Bax is given for control (Lanes 1 and 2) and Vz-treated (Lanes 3–8) tumors. GAPDH served as an internal control. Lanes 1 and 2 contain RNA from control tumors, Lanes 3 and 4 contain RNA from tumors treated with 0.32 mg Vz/kg b.w. for 10 days, Lanes 5 and 6 contain RNA from tumors treated with 2.5 mg Vz/kg b.w. for 4 days, and Lanes 7 and 8 contain RNA from tumors treated with 2.5 mg Vz/kg b.w. for 10 days. In Vz-treated tumors, the expression levels of Bcl-2 are consistently lower than those of Bax. The level of Bax in Vz-treated tumors is equal or lower than that seen in control tumors.

 
Vz Modulates Bcl-2 and Bax Expression in Mammary Tumors.
To test the hypothesis that Bcl-2 and Bax are involved in modulation of ACD in mammary tumors, rats with palpable (8–10 mm) tumors were treated for 4 or 10 days with Vz at 0.32 or 2.5 mg/kg b.w. In a previous study, we found that these doses decreased serum estradiol and increased apoptosis in mammary tumors without severely damaging tissue architecture, at least within the first 10 days of treatment (25) . A comparison between the values of apoptotic cells (AI) and Bcl-2 and Bax expression in individual tumors is given in Table 4Citation . From the results presented, it is evident that: (a) AI in the control tumors is below 1.0%; (b) Bcl-2 and Bax are variably expressed in control tumors, with differences in the scores between 1+ and 3 +; (c) Vz, given for 4 days, increases AI in tumor cells (P < 0.001); (d) a further extension of Vz administration for up to 10 days did not increase AI but rather decreased AI (P < 0.01) as compared with values for animals treated with Vz for 4 days; however, at both time points, the AI remained higher than that seen in the control tumors (P < 0.001); and (e) Vz decreased Bcl-2 but to less extend Bax expression.


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Table 4 Apoptotic cell death (AI), Bcl-2, and Bax expression in mammary tumorsa

 
In Vz-treated tumors, Bcl-2 decreased in the areas where the percentage of apoptotic cells was high (Fig. 1HCitation ). Surprisingly, in apoptotic cells, a high level of Bcl-2 expression was also observed (Fig. 1Citation H, arrow), as revealed by double labeling of apoptotic cells with the TUNEL assay (brown staining of the nucleus) and for Bcl-2 expression by ICH (red staining of the cytoplasm; see "Materials and Methods"). Bax, on the other hand, was highly expressed in the periphery of tumor outgrowths (Fig. 1Citation I, long arrow) as well as in the areas of cell death. In apoptotic cells, an increase in Bax was also found. This was confirmed both by using a low concentration of anti-Bax antibody, which identified cells with a very high level of Bax expression, and by double labeling of apoptotic cells, as indicated above (Fig. 1Citation I, short arrow). In 5 of 20 tumors treated with Vz, Bax was expressed in both the cytoplasm and the nucleus. This was not found for Bcl-2.

The data from ICH for Bcl-2 and Bax expression in Vz-treated tumors were also supported by Western and Northern blot analyses. Eight nonselected tumors were treated for 4 or 8 days with either 0.32 or 2.5 mg Vz/kg b.w. An overall lower level of Bcl-2 expression was found in Vz-treated tumors as compared with control tumors. Selected blots are presented in Fig. 2A and BCitation . Low levels of Bcl-2 expression were observed in both 4- and 10 (Lanes 5 and 7)-day-treated animals. In two other tumors (Lanes 6 and 8), the values of Bcl-2 expression appear close to those of some control tumors (Lane 4). Bax was expressed in all Vz-treated tumors (Lanes 5–8). In two tumors treated for 4 days with Vz, (Lanes 5 and 6), Bax expression was higher than that seen in control tumors. In the other two tumors, the values of Bax were similar to those of control animals. It appears that Bax expression is less affected by Vz than Bcl-2 expression.

The results from Western blot were also supported by Northern blot analysis (Fig. 2BCitation ). The first two lanes contain RNA isolated from control tumors (Lanes 1 and 2). The next six lanes contain RNA isolated from tumors treated with Vz (Lanes 3 and 4, 0.32 mg Vz/kg b.w. for 10 days; Lanes 5 and 6, 2.5 mg Vz/kg b.w. for 4 days; and Lanes 7 and 8, 2.5 mg Vz/kg/b.w. for 10 days). From the Northern blots, it is evident that for all doses and time points of Vz used, the Bcl-2 transcription level is much lower than the Bax transcription level. The lowest RNA level was found in the animals treated for 10 days with 2.5 mg Vz/kg b.w. This was also confirmed by the densitometry data. The average Bcl-2 transcriptional level decreased 56.23% in the group treated with 0.32 mg Vz/kg b.w. for 10 days, decreased 39.0% in the group treated with 2.5 mg Vz/kg b.w. for 4 days, and decreased 55.29% in the group treated with 2.5 mg Vz/kg b.w. for 10 days (Fig. 2BCitation ). In the same tumors, the average Bax transcriptional level decreased 35.6% in the group treated with 0.32 mg Vz/kg b.w. for 10 days, decreased 28.0% in the group treated with 2.5 mg Vz/kg b.w. for 4 days, and decreased 22.39% in the group treated with 2.5 mg Vz/kg b.w. for 10 days. These data indicate that in Vz-treated groups, the percentage of decrease is much higher for Bcl-2 than for Bax and that the alterations appear to be dose dependent.


    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
In this study, we found for the first time that both Bcl-2 and Bax are differentially expressed in epithelial, myoepithelial, and stroma cells of various normal mammary gland structures and that alterations in the expression of both proteins occur in the late, premalignant (CIS), and malignant stages of mammary carcinogenesis. The age of the animals does not appear to be critical for the level of Bcl-2 and Bax expression in both epithelial and myoepithelial cells.

In normal mammary gland structures, Bcl-2 and Bax were equally expressed in the cytoplasm of epithelial cells, regardless of whether they were localized in TEBs, ducts, or alveoli. However, in myoepithelial cells, the level of Bax expression was much high than that of Bcl-2 expression. The differences in Bcl-2 and Bax expression between myoepithelial and epithelial cells were not associated with corresponding differences in ACD, which was very low for both cell types (AI < 1.0%). High-level Bax expression and low-level Bcl-2 expression in myoepithelial cells have also been observed in the mammary gland of BALB/c, transgenic WAP-Bcl-2, and p53-/- mice (32) . Epithelial cells in the mammary gland have been considered sensitive to hormone manipulations, and they usually die after ovariectomy or hormone ablation, whereas myoepithelial cells are resistant to apoptotic stimuli and survive after ovariectomy or hormone ablation (1415) . Thus, our data and those of others on Bcl-2 and Bax expression in mammary epithelial and myoepithelial cells contradict the general dogma assuming Bcl-2 as a cell survival-promoting protein and Bax as a cell death-promoting protein (24) . It appears that the levels of Bcl-2 and Bax expression in various mammary cell populations do not directly reflect their sensitivity to apoptotic stimuli and/or that other members of these families (Bcl-x/L, Bcl-x/S, Bak, and Bad) might be involved in the modulation of cell death signals (561929) . Recently, new members of the Bcl-2 family have been identified, and they have also been variably expressed in various mammary cell populations. For instance, a proapoptotic gene/protein prostate apoptotic response-4 (Par-4) was highly expressed in myoepithelial cells and undetectable in terminally differentiated ductal and alveolar cells. After hormone ablation, Par-4 increased in ductal and alveolar cells, suggesting its involvement in mediating cell death in these cell types (33) .

We also found that in most HLs, the level of Bcl-2 and Bax expression was similar or close to that of normal MECs, suggesting that in the early stages of mammary carcinogenesis, only minor alterations in the expression of both proteins may occur (Table 2)Citation . Because Bcl-2 and Bax were similarly expressed in TEBH, DH, and AH, it appears that the origin of lesions (TEBs, ducts, and alveoli) does not affect the expression of both proteins. Dissociation between Bcl-2 and Bax expression occurred later, in CIS and tumors and particularly in the central tumor areas, where ACD and non-ACD increased sharply (34) . Bcl-2 was expressed mostly in the peripheral (proliferating) tumor areas but decreased in the areas of cell death, contrary to Bax, which was highly expressed in the peripheral 1–2 cell layers (close to the stroma) and remained unchanged or even increased in the areas of cell death. It appears that the decreased Bcl-2:Bax ratio in the central tumor areas may potentate ACD. Surprisingly, we found that in apoptotic cells, both Bcl-2 and Bax are highly expressed, indicating their involvement in the final phase of the apoptotic pathway. The increase of Bcl-2 in apoptotic cells could be the result of conformational changes in the protein or of dimerization with other proteins that may have a similar molecular configuration. Thus, our data and those of others support the theory that a deregulation in the synthesis and/or function of both Bcl-2 and Bax potentates ACD in mammary tumors.

A comparison was also made between the histomorphology of tumors and the levels of Bcl-2 and Bax expression (Table 3)Citation . However, the differences in the expression of both proteins between various histological types were not significant. In some comedocarcinomas, there are many areas of cell death, and the tumor cells surrounding these areas usually showed a low level of Bcl-2 expression and a high level of Bax expression, contrary to adenocarcinomas and papillary carcinomas, in which a central necrosis occurred relatively late in tumor progression. It has been suggested that hypoxia, which is a common phenomenon in solid tumors, may potentate ACD and that both Bcl-2 and Bax participate in this process (35) . Additional tumors, particularly comedocarcinomas and nondifferentiated carcinomas, need to be examined to assess the role of histomorphology and tumor differentiation in Bcl-2 and Bax expression.

To further test the hypothesis that Bcl-2 and Bax are involved in the regulation of cell death in mammary tumors, animals with established tumors (which in this tumor model are ER+) were treated with Vz at doses that suppress mammary carcinogenesis and tumor growth. Vz is a nonsteroidal inhibitor of the activity of aromatase, which is the key enzyme involved in estrogen biosynthesis. A decrease in aromatase activity leads to a decrease in estradiol circulation and tissue levels and thus induces the regression of ER+ mammary tumors (36) . In a recent study, we found that within the first 2–4 days of Vz administration, the number of apoptotic cells increased sharply in mammary tumors, concomitant with a decrease in cell proliferation (25) . Here we observed an overall decrease in Bcl-2 and a reduced effect on Bax (see Table 2Citation and Figs. 1 and 2Citation Citation ), suggesting differential involvement of both molecules in regulating ACD in this tumor model. The consistent decrease in Bcl-2 in the areas of tumor cell death and the lack of a clear effect or elevation of Bax in the same tumor areas indicate a decrease in the Bcl-2:Bax ratio and that the loss of Bcl-2 is more potent than the gain of Bax in regulating ACD in mammary tumors. These data support recent studies on mice with knockout Bcl-2 and/or Bax genes, in which the gain of Bcl-2 has been considered more potent than the loss of Bax in regulating MEC survival (22) .

The dissociation in the levels of Bcl-2 and Bax expression in Vz-treated tumors was also confirmed by Western and Northern blot analyses. Bcl-2 and Bax protein and mRNA levels decreased in almost all tumors examined, and the percentage of decrease was more pronounced for Bcl-2 than for Bax at both Vz dose levels. It appears that Vz-induced inhibition of cell proliferation and induction of cell death in mammary tumors (25) affect the synthesis and function of Bcl-2 and Bax at the mRNA and protein levels. Interestingly, we found that both Bcl-2 and Bax proteins increased in apoptotic cells, suggesting that they may play a dual role in (a) determining the sensitivity of tumor cells to apoptotic stimuli and (b) attending as executors in the final phase of the cell death cascade (2937) . Recent studies from Krajewski et al. (38) have also indicated increased Bcl-2 in neurons undergoing apoptosis after acute cerebral ischemia.

In conclusion, our data indicate that in the course of mammary carcinogenesis, deregulation in the expression of Bcl-2 and Bax occurs in the late, premalignant, and malignant stages of the neoplastic process. We also found that Vz-induced ACD in tumors is primarily associated with decreased Bcl-2 and alterations in the Bcl-2:Bax ratio. It appears that loss of Bcl-2 is a more potent indicator than gain of Bax for the response of mammary tumors to apoptotic stimuli.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 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 with carcinogen. The animals were fed 4% Purina Chow ad libitum and had free access to water. Rats were weighed weekly and checked daily for signs of toxicity. Beginning 3 weeks after MNU administration, the animals were palpated twice weekly to monitor tumor development in mammary gland.

Chemical Carcinogen
MNU was obtained from Ash Stevens, Inc. (Detroit, MI), dissolved in sterile acidified saline (pH 5.0), and injected i.p. (50 mg/kg b.w.) twice, when the animals were 43 and 50 days old. Control animals at the same age received sterile saline only. Two doses of MNU were used to increase the incidence and frequency of mammary gland lesions (39) .

Vz
Vz (R-83842) was obtained from the Janssen Research Foundation (Springfield, PA). A dose of 0.32 or 2.5 mg Vz/kg b.w. was given daily by gavage for 4 or 10 days. Vz administration was initiated when tumors reached 8–12 mm in diameter. In a recent study, we found that Vz at doses in the range of 0.08–2.5 mg Vz/kg b.w. is an efficacious inhibitor of mammary carcinogenesis and tumor growth, and this was associated with a significant decrease in serum estradiol (36) .

Histomorphology of Lesions
To evaluate the origin and frequency of small-sized HLs and CIS, we used the whole mount procedure for processing of mammary glands (40) . Palpable tumors or any abnormal masses in mammary glands were removed and cut in two halves: (a) one half was fixed in 10% neutral formalin for histomorphology, estimation of apoptotic cells, and analysis of Bcl-2 and Bax expression by ICH; and (b) the other half was frozen in liquid nitrogen for Western and Northern blot analyses. CIS and carcinomas were classified by using the criteria suggested by Russo et al. (41) . Various HLs were classified as TEBHs, DHs, and AHs (42) .

Programmed Cell Death (Apoptosis)
Apoptotic cells were identified by the TUNEL method (43) as recommended by the ApopTag in situ hybridization detection kit (Oncor, Gaithersburg, MD). The top sections on each slide, which were incubated without digoxigenin-dUTP, were used as a negative control. As a positive control, we used sections from mammary gland of rats 6 days after ovariectomy (provided by Oncor), where the number of apoptotic cells was high. Tissue sections were counterstained with methyl green for visualization of tumor morphology. From each tumor, more than 2000 cells located within the peripheral zone (5–6 cell layers from the stroma) were evaluated for apoptotic cells (AI).

Expression of Bcl-2 and Bax
ICH.

For ICH, tissue sections were heated for 10 min in 0.01 M citrate buffer in a microwave to increase antigen-antibody binding. For overnight incubation, a 1:1000 dilution of polyclonal Bcl-2 (catalogue number 13456E) and a 1:1500 dilution of polyclonal Bax (catalogue number 13686E) antibodies diluted in 1.0% BSA were used. Both antibodies were obtained from PharMingen (San Diego, CA). In selected tissue sections, two concentrations of Bcl-2 (1:500 and 1:2500) and Bax (1:1000 and 1:5000) were used to identify variability in the expression of both proteins in individual cells. The antigen-antibody complex was detected by the ABC kit (Vector Laboratories, Burlingame, CA). The slides were counterstained with hematoxylin for identification of tissue architecture. The level of expression of both proteins was evaluated by using a semiquantitative scale (0, lack of expression; 1+, weak expression; 2+, medium expression; 3+, strong expression; and 4+, very strong expression) as recommended by Krajewski et al. (44) . Normal ductal and alveolar cells surrounding various mammary lesions and tumors served as a positive control for Bcl-2 and Bax expression. The top sections of each slide, which were not treated with the corresponding antibody, were used as negative controls. The level of expression of both proteins was assessed independently by two cytologists (K. C. and A. S.), and an agreement was achieved in most of the lesions evaluated. If a disagreement was found in the scores of some slides, the samples were reevaluated or restained, and consensus was achieved.

To assess whether Bcl-2 and Bax are expressed in apoptotic cells, selected slides were stained using the TUNEL assay for identification of apoptotic cells, followed by overnight incubation with Bcl-2 or Bax antibodies for visualization of the corresponding proteins. Briefly, after completing the TUNEL assay as indicated above and staining the apoptotic cells with 3,3'-diaminobenzidine, the slides were washed in PBS for 15 min, boiled for 10 min in 0.01 M citrate buffer in a microwave, blocked for 20 min in blocking serum, and left overnight at 4°C with the corresponding antibodies at a concentration of 1:500 for Bcl-2 and 1:1000 for Bax, respectively. The antigen-antibody complex was revealed by the ABC kit, and the slides were stained at the end by red AEC. The, apoptotic cells were easily recognized by the brown staining of the nucleus, whereas Bcl-2 and Bax proteins were identified by the red color of the cytoplasm (see Fig. 1H and ICitation ).

Western Blot Analysis.
For Western blot analysis, the tissue samples were disintegrated by a homogenizer and lysis buffer. Lysates were centrifuged for 10 min at 1000 x g, and the supernatant containing the cytoplasm proteins was precipitated by methanol/chloroform. Protein (10 mg/lane) was electrophoretically separated using 12% SDS-PAGE. Bcl-2 and Bax were identified using the same antibodies used for ICH (Fig. 2ACitation ). After transferring the gels to a nitrocellulose membrane and blocking overnight (3% BSA in PBS), membranes were incubated with the corresponding antiserums diluted 1:2000 (anti-Bax) and 1:3000 (anti-Bcl-2). Thereafter, filters were incubated with alkaline phosphatase-conjugated goat antirabbit antibodies. Bands were visualized by the chemiluminescent substrate nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (DuPont New England Nuclear, Boston, MA). A rabbit antibody against {alpha}-actin was used to control for protein loading. Jurkat cells were used as control cell population that expresses both Bcl-2 and Bax.

Northern Blot Analysis.
Total RNA was isolated with TRI reagent provided by Molecular Research Center, Inc. (Cincinnati, OH). The quantity and purity of the RNA were measured by a spectrophotometer at a wavelength of 260 nm versus 280 nm, and the quality of RNA was checked on the formaldehyde agarose gel. For Northern blot analysis, 20 µg of total RNA were fractionated on a 1.0% agarose-1.7 M formaldehyde gel. The gel was subsequently transferred to a Nylon membrane in 20x SSC [3 M NaCl and 0.3 M sodium citrate (pH 7.0)] by using the Turboblotter Rapid Downward Transfer System provided by Schleicher & Schuell Co. (Keene, NH). The membrane was then baked for 2 h at 80°C in a vacuum oven. Fragments of Bcl-2, Bax, and GAPDH transcripts were PCR amplified and purified by using the Concer Rapid Gel Extraction System provided by Life Technologies, Inc. (Grand Island, NY). Purified probes were labeled by using the Multiprime DNA Labeling System (Amersham Pharmacia Biotech). Prehybridization was performed at 42°C in 50% formamide, 6x SSC, 5x Denhardt’s reagent, 0.5% SDS, and 200 µg/ml low molecular weight heterologous DNA for 2 h. Hybridization was carried out at 42°C under the same conditions as prehybridization for 18 h. After hybridization, the membrane was washed twice briefly in 2x SSC and 0.1% SDS at room temperature and then washed three times in 0.1x SSC and 0.1% SDS for 15 min at 68°C. The membrane then was exposed to X-ray film at -70°C with an intensifying screen or to a phosphor imager screen. After each use, the membrane was stripped and reused. Bcl-2, Bax, and GAPDH probes were hybridized to the same membrane (Fig. 2BCitation ).

Statistical Analysis
Differences between control and treated groups as well as between various parameters were evaluated by the {chi}2 test and Fisher’s two-tailed t test.


    Acknowledgments
 
We thank Kevin Grandfield for editing the manuscript.


    Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by National Cancer Institute Grants NO1-CN 55179-MAO and N01-CN-65123-MAO (to K. C.). Back

2 To whom requests for reprints should be addressed, at Department of Surgical Oncology, University of Illinois at Chicago, 840 South Wood Street (M/C 820), Chicago, Illinois 60612. Phone: (312) 996-5347; Fax: (312) 996-9365; E-mail: Christov{at}UIC.edu Back

3 The abbreviations used are: ACD, apoptotic cell death; Vz, vorozole; TEB, terminal end bud; TEBH, terminal end bud hyperplasia; CIS, carcinoma in situ; ER, estrogen receptor; MNU, methylnitrosourea; MEC, mammary epithelial cell; ICH, immunocytochemistry; AI, apoptotic index; DH, ductal hyperplasia; AH, alveolar hyperplasia; HL, hyperplastic lesion; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labeling; b.w., body weight; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Back

Received for publication 8/25/99. Revision received 5/10/00. Accepted for publication 5/31/00.


    References
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

  1. Wyllie A. H. Apoptosis and the regulation of cell numbers in normal and neoplastic disease: an overview. Cancer Metastasis Rev., 11: 95-103, 1992.[Medline]
  2. Evan G., Litlewood T. A matter of life and cell death. Science (Washington DC), 281: 1317-1322, 1998.[Abstract/Free Full Text]
  3. Hockenbery D. M., Zutter M., Hickey W., Nahm M., Korsmeyer S. J. Bcl-2 protein is topographically restricted in tissues characterized by apoptotic cell death. Proc. Natl. Acad. Sci. USA, 88: 6961-6965, 1991.[Abstract/Free Full Text]
  4. Oltvai Z. N., Milliman C. L., Korsmayer S. J. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell, 74: 609-619, 1993.[Medline]
  5. Yang, E., Zha, J., Jokel, J., Boise, L. H, Thompson, C. B, and Korsmayer, S. J. Bad, a heterodimeric partner for Bcl-XL and bcl-2, displaces Bax and promotes cell death. Cell, 80: 285–291, 1995.
  6. Adams J. M., Cory S. The Bcl-2 protein family: arbitors of cell survival. Science (Washington DC), 281: 1322-1326, 1998.[Abstract/Free Full Text]
  7. Russel D. L., Kaklamanis L., Pezzella F., Gatter K. C., Harris A. L. Bcl-2 in normal breast and carcinoma, association with ER positive, epidermal growth factor negative tumors and in situ cancer. Br. J. Cancer, 69: 135-139, 1994.[Medline]
  8. Binder C., Marx D., Binder L., Schour A., Hiddemann W. Expression of Bax in relation to Bcl-2 and other predictive parameters in breast cancer. Ann. Oncol., 7: 129-133, 1996.[Abstract/Free Full Text]
  9. Bhargava V., Kell D. L., Rijan M. V., Warnke R. A. Bcl-2 immunoreactivity in breast carcinoma correlates with hormone receptor positivity. Am. J. Pathol., 145: 535-540, 1994.[Medline]
  10. Bargou R., Daniel P., Mapara M. Y., Bommert K., Wagener C., Kallinich B., Royer H. D., Durken B. Expression of the Bcl-2 gene family in normal and malignant breast tissues: low bax-{alpha} expression in tumor cells correlates with resistance towards apoptosis. Int. J. Cancer, 60: 854-859, 1995.[Medline]
  11. Bargou R. C., Wagner C., Bommert K., Mapara M. Y., Daniel P. T., Arnold W., Dietel M., Guski H., Feller A., Royer H. D., Dorken B. Overexpression of death promoting gene Bax-{alpha} which is down-regulated in breast cancer restores sensitivity to different apoptotic stimuli and reduces tumor growth in SCID mice. J. Clin. Invest., 97: 2651-2659, 1996.[Medline]
  12. Veronese S., Mauri F. A., Caffo O., Scaioli M., Aldovini D., et al Bax immunohistochemical expression in breast carcinoma: a study with long follow-u. p. Int. J. Cancer, 79: 13-18, 1998.[Medline]
  13. Krajewski S., Blomquist C., Francila K., Krajewska M., Wasenius V. M., Niskanen E., Nordling S., Reed J. C. Reduced expression in proapoptotic gene Bax is associated with poor response rates to combination chemotherapy and shorter survival in women with metastatic breast adenocarcinoma. Cancer Res., 55: 4471-4478, 1995.[Abstract/Free Full Text]
  14. Walker N. I., Bennett R. E., Kerr J. F. R. Cell death by apoptosis during involution of the lactating breast in mice and rats. Am. J. Anat., 185: 19-32, 1989.[Medline]
  15. Furth P. A., Bar-Peled U., Li M. Apoptosis and mammary gland involution; reviewing the process. Apoptosis, 2: 19-24, 1997.[Medline]
  16. Strange R., Li F., Saurer S., Burkhardt A., Fries R. R. Apoptotic cell death and tissue remodeling during mouse mammary gland involution. Development (Camb.), 115: 49-58, 1992.[Abstract]
  17. Lund L. R., Romer J., Thomasset N., Solberg H., Pyke C., Bissell M. J., Dano K., Werb Z. Two distinct phases of apoptosis in mammary gland involution: proteinase independent and dependent pathways. Development (Camb.), 122: 181-193, 1996.[Abstract]
  18. Li M., Liu X., Robinson G., Bar-Peled U., Wagner K. U., Young W. S., Henninghousen L., Furth P. A. Mammary-derived signals activate programmed cell death during the first stage of mammary gland involution. Proc. Natl. Acad. Sci. USA, 94: 3425-3430, 1997.[Abstract/Free Full Text]
  19. Heermeier K., Benedickt M., Li M., Furth P. A., Nunez G., Henninghousen L. Bax and Bcl-xs are induced at the onset of mammary involution. Mech. Dev., 56: 197-207, 1996.[Medline]
  20. Jager R., Herzer U., Schenkel J., Weiher H. Overexpression of Bcl-2 inhibits alveolar cell apoptosis during involution and accelerates c-myc induced tumorigenesis of the mammary gland of transgenic mice. Oncogene, 15: 1787-1795, 1997.[Medline]
  21. Li M., Hu J., Heermeier K., Henninghousen L., Furth P. A. Apoptosis and remodeling of mammary gland tissue during involution proceed through p53-independent pathways. Cell Growth Differ., 7: 13-20, 1996.[Abstract]
  22. Schorr K., Li M., Bar-Peled U., Lewis A., Heredia A., Lewis B., Knudson M., Korsmeyer S. J., Jager R., Weiher H., Furth P. A. Gain of Bcl-2 is more potent than Bax in regulating mammary epithelial cell survival in vivo. Cancer Res., 59: 2541-2545, 1999.[Abstract/Free Full Text]
  23. Witty J. P., Lempka T., Coffey R. J., Jr.,, Matrisian L. M. Decreased tumor formation in 7,12-dimethylbenzanthracene-treated stromelysin-1 transgenic mice is associated with alterations in mammary epithelial cell apoptosis. Cancer Res., 55: 1401-1406, 1995.[Abstract/Free Full Text]
  24. Srivastava P., Russo J., Russo I. H. Chorionic gonadotropin inhibits rat mammary carcinogenesis through activation of programmed cell death. Carcinogenesis (Lond.), 18: 1799-1808, 1997.[Abstract/Free Full Text]
  25. Christov, K., Green, A., Shilkaitis, A., Mehta, R. G., Grubbs, C., Kelloff, G., and Lubet, R. Cellular mechanisms of response of mammary tumors to aromatase inhibitors. Effects of Vorozole. Breast Cancer Res. Treat. 60: 117–128, 2000.
  26. Hickman J. A. Apoptosis induced by anti-cancer drugs. Cancer Metastasis Rev., 11: 121-139, 1992.[Medline]
  27. Thompson C. B. Apoptosis in pathogenesis and treatment of disease. Science (Washington DC), 267: 1456-1462, 1995.[Abstract/Free Full Text]
  28. Strobel T., Kraeft S. K., Chen L. B., Cannistra S. A. Bax expression is associated with enhanced intracellular accumulation of paclitaxel: a novel role for Bax during chemotherapy-induced cell death. Cancer Res., 58: 4776-4781, 1998.[Abstract/Free Full Text]
  29. Knudson C. M., Korsmeyer S. J. Bcl-2 and Bax function independently to regulate cell death. Nat. Genet., 1916: 358-363, 1997.
  30. Gulliano P. M., Pettigrew H. M., Grantham F. H. N-Nitrosomethylurea as mammary gland carcinogen in rats. J. Natl. Cancer Inst., 54: 401-414, 1975.
  31. Russo J., Gusterson B. A., Rogers A. E., Russo I. H., Wellings S. R., Zwieten M. J. Comparative study on human and rat mammary tumorigenesis. Lab. Investig., 62: 244-278, 1990.[Medline]
  32. Humphreys R. C., Krajewska M., Krnacik S., Jaeger R., Weihert H., Krajewski S., Reed J. C., Rosen J. M. Apoptosis in the terminal end bud of the murine mammary gland: a mechanism of ductal morphogenesis. Development (Camb.), 122: 4013-4022, 1996.[Abstract]
  33. ogheart, E. R., Sells, S. F., Walid, A. J., Malone, P., Williams, N. M, Weinstein, M. H., Strange, R., and Rangnekar, V. M. Immunohistochemical analysis of proapoptotic protein Par-4 in normal rat tissues. Cell Growth Differ., 8: 881–890, 1997.
  34. Christov K., Swanson S., Guzman R., Thordarson G., Talamantes F., Nandi S. Cell proliferation and apoptosis during mammary carcinogenesis in pituitary isografted mice. Carcinogenesis (Lond.), 17: 1741-1746, 1996.[Abstract/Free Full Text]
  35. Graeber T. G., Osmanlan C., Jacks T., Housman D., Koch C. J., Lowe S. W., Glaccla A. J. Hypoxia-mediated selection of cells with diminished apoptotic potential in solid tumors. Nature (Lond.), 379: 88-92, 1996.[Medline]
  36. Lubet R. A., Steele V. E., DeCoster R., Bouden C., You M., Juliana M. M., Eto I., Kelloff G. J., Grubbs C. J. Chemopreventive effects of aromatase inhibitor vorozole (R83842) in the methylnitrosurea-induced mammary cancer model. Carcinogenesis (Lond.), 19: 1345-1351, 1998.[Abstract/Free Full Text]
  37. Askhenazi A., Dixit V. M. Death receptors: signaling and modulation. Science (Washington DC), 281: 1305-1308, 1998.[Abstract/Free Full Text]
  38. Krajewski S., Mai J. K., Krajewska M., Sikorska M., Mossakowski M. J., Reed J. C. Up-regulation of Bax protein levels in neurons following cerebral ischemia. J. Neurosci., 15: 6364-6376, 1995.[Abstract/Free Full Text]
  39. Grubbs C. J., Peckham J. C., Cato K. D. Mammary carcinogenesis in rats in relation to age at time of N-nitroso-M-methylurea administration. J. Natl. Cancer Inst., 70: 209-212, 1983.
  40. Medina D. , Preneoplastic lesions in mouse mammary tumorigenesis Bush H. eds. . Methods in Cancer Research, 7: 3-53, Academic Press 1978.
  41. Russo J., Russo I. H., van Zwieten M. J., Rogers A. E., Gusterson B. Classification of neoplastic and non-neoplastic lesions of the rat mammary gland Jones T. C. Mohr U. Hunt R. D. eds. . Integument and Mammary Glands, Monographs on Pathology of Laboratory Animals, : 275-340, Springer Verlag New York 1989.
  42. Anderson C. H., Hussain R. A., Han M. C., Beattie C. W. Estrus cycle dependence of nitrosomethylurea (MNU)-induced preneoplastic lesions in mammary gland. Cancer Lett., 56: 77-84, 1991.[Medline]
  43. Gavrielli Y., Sherman Y., Ben-Sasson S. A. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J. Cell Biol., 119: 493-501, 1992.[Abstract/Free Full Text]
  44. Krajewski S., Krajewska M., Shabak A., Myashita T., Wang H. G., Reed J. C. Immunocytochemical determination of in vivo distribution of Bax, a dominant inhibitor of Bcl-2. Am. J. Pathol., 145: 1323-1336, 1994.[Medline]



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