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| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cell Growth & Differentiation |
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.]
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Introduction |
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TOPAbstractIntroductionResultsDiscussionMaterials and MethodsReferences |
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, 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- 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.
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Results |
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).
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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. 1A
). 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. 1B
,
arrow; score, 4+) and showed medium or strong
expression in body cells, fibroblasts, and endothelial cells (scores,
2+ and 3+; Fig. 1B
). 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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. 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)
.
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. 1C
), whereas CIS (Fig. 1D
) and carcinomas had low
(1+), medium (2+), or high (3+) levels of Bcl-2 and Bax expression
(Table 1)
. Myoepithelial cells surrounding various lesions and tumors
had very high (4+) Bax expression and low (1+) or medium (2+) Bcl-2
expression (Fig. 1
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 2
. No significant difference in Bcl-2 and Bax expression was found
between normal MECs and HLs (Table 2)
: 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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). 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)
. In most tumors, independent of
their histology, scores of 2+ and 3+ predominated. Few tumors had
scores of 1+ or 4+ (Table 3)
. 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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, 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. 1
G, arrow).
The variability in Bcl-2 and Bax expression in tumors was also
confirmed by Western blot analysis (Fig. 2
). Eight nonselected control tumors (the values of four are presented in
Fig. 2A
, Lanes 1–4) and eight Vz-treated tumors
(the values of four are presented in Fig. 2A
, 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. 2A
,
Lanes 5–8).
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. 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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). Surprisingly, in
apoptotic cells, a high level of Bcl-2 expression was also observed
(Fig. 1
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. 1
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. 1
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 B
. 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. 2B
). 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. 2B
). 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.
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Discussion |
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TOPAbstractIntroductionResultsDiscussionMaterials and MethodsReferences |
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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)
. 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)
. 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 2
and Figs. 1 and 2
), 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.
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Materials and Methods |
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TOPAbstractIntroductionResultsDiscussionMaterials and MethodsReferences |
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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 I
).
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. 2A
). 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 -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. 2B
).
Statistical Analysis
Differences between control and treated groups as well as
between various parameters were evaluated by the
2 test and Fisher’s two-tailed t
test.
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Acknowledgments |
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Footnotes |
|---|
1 Supported by National Cancer Institute Grants
NO1-CN 55179-MAO and N01-CN-65123-MAO (to K. C.). 
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
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.
Received for publication 8/25/99. Revision received 5/10/00. Accepted for publication 5/31/00.
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References |
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TOPAbstractIntroductionResultsDiscussionMaterials and MethodsReferences |
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expression in tumor cells correlates with resistance towards apoptosis. Int. J. Cancer, 60: 854-859, 1995.[Medline]
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]
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