Expression of p53, bcl-2, bax and bel-xi in ductal carcinoma in situ (DCIS) of the breast

Expression of p53, bcl-2, bax and bel-xi in ductal  carcinoma in situ (DCIS) of the breast

AmaiN Ahmad,a MD; Eman MS Muhammad,b MD, PhD;

Asem Elsani MAli, c MD

a) Department of Pathology, Sohag Faculty of Medicine, Egypt. b) Department of Pathology, Sohag Faculty of Medicine, Egypt. c) Department of Surgery, Sohag Faculty of Medicine, Egypt.

Background: DCIS is a malignant proliferation of mammary ductal epithelial cells without invasion beyond the basementmembrane. Bcl-2, bax, bel-xiand p53areimplicatedin regulation of apoptosis. This study aimed to clarify the role of these gene products in DCIC.

Patients and methods:  Immunohistochemical staining of 13 cases of DCIS with bcl-2, bax, bel-xi and p53 was performed.

Results:  DCIS was graded into;  42% of low, 16% of intermediate,  and 42% of high grade cases. P53 was negative in all low grades,  positive in 66.7% intermediate grade, and in all high grade  DCIS. Bcl-2  was positive in all DCIS with variable intensities.  Bax was positive in 80% of low grade,  100% of intermediate grade,  and in 100% of high grade DCIS. Bel-xi was positive in  80% of low grade,  and in  all intermediate and high grade  DCIS.  P53 was inversely correlated with bcl-2 expression in DCIS (p<0.02). Insignificant positive correlation was present between p53 and bax expression and between p53 and bel-xi expression in DCIS. An inverse correlation was present between bcl-2 and bel-xi expression and between bcl-2 and bax expression in DCIS (p<0.01  for each). Positive correlation was present between bax and bel-xi expression (p<O.000) in DCIS.

Conclusions: p53 mutation is an early event in the evolution of breast cancer and its expression correlates  with high grade lesions.  Strong  positive  correlation between  bax  and bel-xi, but the prognostic value of bcl-2 protein family in DCIC is still in need to more studies to be clarified.

Key words: Ductal carcinoma in situ, bcl-2 bax, bel-xi and p53.

Abbreviations:  Ductal carcinoma in situ (DCIS), carcinoma in situ (CIS), invasive carcinoma (IC), inhibitor  of apoptosis proteins (lAP), Hematoxylin and Eosin (H&E), catalogue number (Cat#), phosphate buffered solution (PBS), staining intensity (Sf), immunohistochemical scores

(IHCS), percentage of positive cells (PP), atypical ductal hyperplasia  (ADH).

Introduction:

The   risk   of   breast   cancer   and   death due to breast cancer are clearly increasing worldwide.! Carcinoma  in situ of the breast (CIS) comprises a heterogeneous group of lesions, covering a wide spectrum of clinical conditions   and   histopathological   changes. With respect to biological behavior, CIS range from biologically aggressive lesions with a substantial  risk of progression  into  invasive

carcinoma (IC), to lesions with a very low malignant   potential.    Previous   studies   of CIS indicate that approximately  a third  will subsequently develop IC. Autopsy studies indicate that CIS is frequently  occurring and it was estimated that about 20% of all women will develop CIS during lifetime. 2

Programmed     cell     death     (apoptosis) may   play   a  role   in  tumor   development and  progression.   Dys-regulation   of  genes

controlling apoptosis may cause mammary cells to become immortal and constitute a cancer.3 Programmed cell death is controlled by  inhibitor   of  apoptosis   proteins   (lAP), which  include  bcl-2  family  members  that either  promote  (ie.,  Bax,  Bcl-xS,  Bad  and Bid)  or  counteract   (ie.,Bcl-2,   Bcl-xL  and Mcl-1) the signals that initiate apoptotic cell death.4

In this study, we aimed to evaluate the expression  of apoptosis related proteins, bcl-

2,  bax,  bcl-xl  and  p53  protein  in DCIS  in

relation  to  its grade.  Furthermore,  we tried to evaluate the possible correlations between these proteins which may have an impact on prognosis of DCIS and its progression to IC.

Patients and methods:

This study was done on 13 retrograde formalin-fixed paraffin-embedded specimens of DCIS of the breast. These specimens were obtained  by lumpectomy, incisional  or Tru­ cut  biopsies  of  breast  masses  which  were sent to Histopathology Laboratory of Sohag University  Hospital  in the  period  between

2001-  2005.  The  diagnosis   of  DCIS  was

established  by examination  of  Hematoxylin and Eosin (H&E) stained sections.

DCIS was classified into three grades according to the criteria of Holland et al (1994),5 into well-differentiated (grade I), moderately  differentiated  (grade II), and poorly differentiated DCIS (grade III). In specimens showing more than one histological grade, DCIS was graded according to the highest grade.

Immunohistochemical staining  for  all cases  with  antibodies  for  bcl-2,  bax,  bel­ xi and p53 was performed using immuno­ peroxidase technique.  Sections were pre­ treated  by  boiling  for  9-15  min  in  citrate buffer (pH 6.0). Incubation with the diluted primary antibody with 1% blocking anti-goat serum was applied for 1-2 hours. Antibodies used were mouse monoclonal antibodies for bcl-2 (Bcl-2 Ab 1, clone 100/D5, Catalogue number; Cat # MS123-PO, LabVision), bax (Bax  Ab1, clone  2D2,  Cat  # MS  -771-PO,

LabVision),  bcl-xl (Bcl-xl  Ab2, clone  7D9, Cat # MS -1334-P1,  LabVision) and rabbit monoclonal antibody for p53 (clone Y5, Cat

#  RM-   2103-R7,   LabVision)   at  dilutions

of 1/100 for bcl-2, bax and bcl-xl and 1/25 for  p53.  The sections  were then  incubated with the biotinylated secondary antibody (Ultravision plus large volume detection system, anti-polyvalent,  HRP, Cat # TP-060- HLX, LabVision) for 15 min. All dilutions were made in phosphate buffered solution (PBS), at pH 7.2, and all of the incubations were   performed   in   humid   chambers    at room temperature.  Between  each two  steps in the staining procedures (except before incubation with the primary antibody), the slides were rinsed three times in PBS. Finally, the sections were lightly counterstained in Mayer's Hematoxylin and mounted on glass slides using DPX (BDH Ltd, Poole, United Kingdom).

The   immunostaining   of   bcl-2,   bcl-xL,

bax, and p53 was analyzed and evaluated  in

10 different tumor fields. For p53, nuclear staining only was considered.6  Expression of

5% of tumor cells for the stain was considered as the threshold  of positive  staining,  as the presence of more than 5% immunoreactivity may   be   associated   with   p53   mutations. 7

For  bcl-2, bax, and bcl-xl tumor  cells were

considered   positive   when   they   displayed a distinct micropunctate golden yellow cytoplasmic staining.

The mean percentage of tumor cells with positive  staining  was  evaluated  and scored as: 0 for < 5%, 1 for 5-25%, 2 for 25-50, 3 for

50-75, and 4 for >75 following  Hussein et al

(2004)8 and Baltaziak et al (2006).9

Staining intensity (SI) was also considered in the evaluation of the immunohistochemical expression   of  p53,  bcl-2,  bax,  and  bcl-xl. It is scored  as; 1 for  weak,  2 for medium,

3 for  intense  staining  following  Hussein  et al (2004).8 Immunohistochemical scores (IHCS) were calculated by multiplying the percentage of positive cells (PP) with the staining intensity (SI). Validation of this method has been described elsewhere.lO

Statistical analysis:

Chi-Square and Pearson's Correlation Coefficient tests were used to evaluate statistical significance of various markers in relation to the grade of DCIS, and in relation to each other, with a statistical significance of p <0.05 for Chi-square test and p <0.02 for Pearson's Correlation Coefficient test.

Results:

Relations   between   the   estimated biological markers in DCIS and tumor grade: Tables (1-5)  and Figures (1-2)  illustrate  the results  of  immunohistochemical  expression of p53, bcl-2, bax and bcl-xl in DCIS of the breast.  DCIS  was  graded  into;  5/13 (42%) low,  3/13   (16%)   intermediate,   and   5/13 (42%)  high  grade cases.  Expression of  p53 was negative in all low grades, positive in 2/3 (66.7%) intermediate grade, and in all high grade 5/5 (100%)  DCIS. Bcl-2 was positive in all cases of DCIS with variable intensities. Bax and Bcl-xl were positive in 4/5 (80%) of low grade, and all (100%) intermediate and high grade DCIS.

Relations between the estimated biological

markers  in  DCIS  of the  breast:  Figure (3) illustrates the correlations between the studied markers in DCIS of the breast. Statistically inverse relationship was present between p53 and  bcl-2  expression  in  DCIS  (r = -0.636, p < 0.02).  Statistically  insignificant  positive relationship  was  present  between  p53  and bax expression and between p53 and bcl-xl expression  in  DCIS (r = 0.524,  p < 0.07  for each). Significant inverse correlation was present between bcl-2 and bcl-xl expression and   between   bcl-2   and   bax   expression in   DCIS   (r = -0.659,   p < 0.01   for   each). Significant positive correlation was present between  bax and  bcl-xl  expression  (r = 1.0, p < 0.000) in DCIS.

Discussion:

It is widely held that breast cancer initiates as the pre-malignant stage of atypical ductal hyperplasia (ADH), progresses into the pre­ invasive   stage   of   DCIS,   and   culminates in IC.11 In the current  study  we tried to investigate       the        immunohistochemical

expression  of some  important  apoptosis related gene products namely; bcl-2, bax, and bcl-xl  proteins  and  tumor  suppressor  gene p53; in 15 cases ofbreast in situ carcinoma in relation to tumor grade. We also tried to find possible relationships between expressions of these proteins in DCIS.

Our  study showed that, p53 is expressed

in areas of in situ carcinoma in 7/13 (48%), and it's expression correlated positively with higher tumor grade (p < 0.004) in agreement with  Mao  et  al,  (2010),12  who  found  that the  frequency   of  p53  missense  mutations was significantly different among the three overall  histological  grade  categories  of DCIS; in 0/49 (0%) of low, 1/23 (4.35%) of intermediate, and 9/22 (40.9%) ofhigh-grade DCIS   (P < .0001).   This   finding   indicated that p53 mutations usually occur before invasion  during  breast  cancer  progression, and that  p53 protein  expression  in DCIS is an important indicator of cancer invasiveness and prognosis of DCIS.13

In agreement with Kayaselcuk et al, (2004),14 bcl-2 expression was not correlated with  the   grade  of  DCIS,  which  suggests that alterations in bcl-2 expression in breast carcinoma are more prominent in invasive rather than in in situ lesions.

In     agreement     with     Rehman     et     al,

(2000),15  and  Kayaselcuk   et  al,  (2004),14 who   found   that   bax   expression   did  not correlate with increasing  histological  grades of DCIS, we found  insignificant  correlation between   bax  expression   and  tumor   grade of        DCIS.   Contradictory   findings        were obtained  by Kapucuoglu et al, (1997)16 and Anagnostopoulos  et al, (2007),17 who found that  bax  protein  expression   in  DCIS  was related  to  more aggressive  neoplasms.  Bel­ xi  expression  did  not  correlated  to  tumor grade of DCIS (p<0.3), in concordance with Kayaselcuk  et  al, (2004).14 This  finding  is contradictory  to  the  findings  of  Fernandez et al, (2000),18 and Espanfia, et al, (2005)15 who   found   positive        correlation   between bcl-xl  expression  and grade  of  DCIS.  This controversy reflects the complexity ofbax and bcl-xl regulation and the need for more studies for these genes on both immunohistochemical

and molecular levels.

Bcl-2 is one member  of a gene family, products  of which are involved in either inhibition  or  promotion   of  cell  death.20  A large  number  of  bcl-2  related  proteins  have been isolated. Protein products of this gene family  share  two  highly  conserved  domains BH 1 & BH 2.21 It is divided  into 3 categories (Tsujimoto and  Shimizu,  2000):  1. Anti­ apoptotic  members  such   as  bcl-2,   bcl-xl, bcl-w  & Mcl-1  and  all  of  which  exerts  an anti-cell death activity and share sequence homology,  particularly  within   the  four regions  BH 1 through  BH 4. 2. Pro-apoptotic members   such  as  bax,  bak  and  bad,  which share  sequence homology in BH1, BH2 and BH3 butnotBH4. 3. BH3 onlyproteins, which include bik, bid and him and share sequence homology only  in  BH3.  They  interact  with bcl-2 and antagonize its survival function.

One ofthe unique features  ofbcl-2 family is their  ability  to  physiologically bind  each other  forming a complex network  of  homo­ and/or heterodimers. 22  The  above  domains BH1  and  BH2  regulate  heterodimerization, and  regulation of cell  death  by members  of this gene family may be achieved through competing  dimerization.23 They  regulate apoptosis  in   a   rheostatic  manner:    in   an excess of bax, for instance, bax homodimers predominate    which     favors     apoptosis.24

Conversely,  in  an  excess   of  bcl-2,   bcl-2/

bax  heterodimers are formed, which  lead  to inhibition of apoptosis. Competition between family  members  also  has  an  effect.  Bcl-xL for  example, inhibits  and  sequestering bax. By binding  bcl-2 and bcl-xL, bad on the other hand, releases bax, which leads to bax homo­ dimerization and promotion  of apoptosis.25

Many members  of the bcl-2 family  such as

bcl-2, bcl-xL and bax, are resident proteins  of the  mitochondrial  membranes, endoplasmic reticulum  and   nuclear   envelope   in  which they  are inserted  via their  carboxyl  terminal ends.  In the  mitochondria, they  form  pores and  act  as  ion  channels.  This  is  probably the   key   to   their    function    in   apoptosis. Namely   induction  of   apoptosis  is  almost invariably accompanied by disruption of the mitochondrial  transmembrane  potential  and

release   of  caspase-  activating  substances, such as cytochrome C and AIF, from the mitochondria. 25

As  the   expression  of  this   gene   family seems  to  be  differentially regulated among cell  types  and  their  stage  of  differentiation, so   biological  impact   of  any   bcl-2   family member  on a cell  is dependent on the  level, selective expression, and dimerization status ofthe gene family_26,27

The death antagonistic and death promoting members  of the bcl-2 family function as potential  oncogenes and tumor  suppressor genes  respectively. The  Darwinian selection of cancer cells by adverse intrinsic conditions (limited  trophic  supply, oxygen shortage), and/or therapeutic agents (chemotherapy, radiotherapy) may favor  survival  of the proliferating cells, which overexpress antiapoptotic gene products.28

We     found      an     inverse      relationship between p53 and bcl-2 which is statistically significant   (p < 0.02)    in   agreement   with several  authors.29-31 Interestingly, it has been demonstrated that in human breast carcinoma cell  lines,  a  mutated   and/or  wild-type p53 down-regulates bcl-2 expression.32

Although p53 is a direct transcriptional activator ofbax,33,34 statistically insignificant relationship was present between p53 and bax expression in DCIS of the breast in agreement with Veronese  et al, (1997),35 Rehman  et al,

(2000),36 and Baccouche et al, (2003).37 This

indicates that  regulation of  bax  is complex, and  may  be  explained  by  the  presence   of p53  mutations  which   affect   its  interaction with  bax protein,  or by intervention of other molecules which  may modulate  the function of either  or both proteins.36

In the  current  study,  negative  correlation

between  bax  and  bcl-2  expression was detected in DCIS  (p < 0.01),  which  concurs with  Kapucuoglu et al, (1997),38 who found that while both proteins  were expressed at the same time in normal and benign epithelium, different  staining patterns  were  observed according to the  degree  of differentiation of the neoplastic epithelium.

In this  study  inverse  correlation was present  between  bcl-2  and bcl-xl  expression

A

LG                       TG      FIG

:..... .

0 '

= ·

::2 :

J..- - - r!fl'

D

LG                   IG                      IIG

Figure {1): A. P53, B. Bel-2, C. Bax and D. Bel-xiexpression in DCIS according to tumor grade.

Figure {2): A. Strong p53, B. weak bel-2,  C. Strong bax and D. Strong bel-xi expression in comedo (.4 & B) and cribriform (C &D) DCIS of the breast. Magnifications: X 200 (A & B) and X400(C&D).

in DCIS of the breast (p < 0.01). This finding can be explained by that although  bcl-2 and bcl-xl  are  homologues  and  both  of  them encode membrane-associated proteins that protect neoplastic  cells fi:om DNA damage­ induced   apoptosis,   differential   expression and regulation ofbcl-2 and bcl-xl in different tumors is present.39

Although  bax  gene  is  proapoptotic  and bcl-xl is antiapoptotic, highly significant positive correlation was present between bax expression  and bcl-xl expression (p < 0.000) in DCIS. This is in agreement  with Guo et al, (2002),40 Baltaziak  et  al, (2006),44 and

Wincewicz et al, (2007),42 who found a positive conelation between bcl-xl and baxin several types of malignant tumors. This result could  be due to possible heteroclimerization of bax and bcl-x1,43,44 which may affect the expression of both or either proteins.

We conclude that p53 mutation is an early event in evolution of breast cancer and its expression conelates with high grade lesions. In addition, it appears that the relation between bcl-2 family members is too complex, with strong  positive conelation between bax and bcl-xl, and that their prognostic value is less important in DCIC of the breast than in IC.

A                                              B

"000

,.00

110.00

<WI.OO

I f    101'0                                                                                                                         •n:10.00

20.00

1000

IO.DO

000                                                                                                                                         0.00

,,{0                  s.oo           1.SO                 tOOO                                                       O.AIO              :2 110              6.00               1.60              10.00

Bcl2

c                              D

11000

<11100

•n :»oo

31100

CJ

=...

7000

1000

1000

000

o.oo              '2.«»              600              7 so        10.00                                                   aoo               ,...    6110                ,..,        1000

Bcl'd                                                       Bclxl

E                                      F

10000

®.00

7600

>000                                                                                                                              ::s....

CJ                                                                                              CJ

=                              =2400

10.00

0.00             2 QI             6.00              '1.60              !000

o.oo

o.oo                 2JIO                Goo       7,60              to ,oo

Bax                                                       Bax

Figure {3): Relation between A. P53 and bel-2, B. P53 and bax, C. P53 and bel-xi, D. bel-2 and bel-xi, E. bel-2 and bax, and bax and bel-xi in DCIS of the breast.

Refea·ence:

1-   Potter   PN:  Westemizing"  women's  1isks?

Breast  cancer  in  lower-income   cmmtlies.

Engl1Med2008;358: 213-216.

2-    Ottesen   G:   Carcinoma    in   situ   of   the female         breast.     A    clinico-pathological, immunohistological, and DNA ploidy study. APMIS Suppl2003; 23: 1--67.

3-    Moreno A, Figueras A, Lloveras B, Escobedo

A. Gliera  E, Siena  A. Fabra  A: Apoptosis

4-    Kayaselcuk   F,  Nmsal  T,  Polat  A. Noyan T, Yildhim  S, Tatim  A. Seydioglu G: Expression  of Survivin,  bcl-2, p53 and ba,'{ in breast cru·cinoma and ductal intraepithelial

neoplasia (DIN  la). 1ExpCiln Cancer Res.,

2004; 23: 105-112

5-    Holland  R,  Peterse  JL,  Millis  RR:  Ductal cru·cinoma in  situ:  A  proposal  for  a  new classification. Semln Dlagn Patholl994; 11:

167-180.

Table (1): P53 expression in DCIS according to tumor  grade.

Table (2): Bcl-2 expression in DCIS according to tumor grade.

Table (3):  Bax expression in DCIS according to tumor  grade.

Table (4): Bcl-xl expression in DCIS according to tumor  grade.

Table  (5):  Relations   between   the   investigated   apoptosis  markers   m DCIS   (Pearson's correlation coefficient; r value).

N,  van  Leeuwen   I,  Vasen  HFA,  Cleton­ Jansen AM, Kraan J, Houwing-Duistermaat JJ, Morreau H, Comelisse  CJ, Devilee P: Characterization of familial non-BRCAl/2 breast tumors by loss of heterozygosity  and immunophenotyping. Clin Cancer Res 2006;

12: 1693-1700.

7-    Wikonkal NM,  Berg RG, van Haselen  CW, Horkay I, Remenyik  E, Begany A, Hunyadi J, van  Vloten  WA, de Gruijl FR: Bcl-2 vs p53  protein  expression  and  apoptotic  rate in human nonmelanoma skin cancers. Arch Dermatol1997; 133: 599--602.

Hussein   MR,  Al-Badaiwy   ZH,  Georgis   MN:

Analysis of p53 and bcl-2 protein expression in  the  non-tumourigenic,   pretumourigenic and tumourigenic keratinocytic hyperproliferative    lesions.   1 Cut  Pathol

2004; 244: 1-9.

8-    Baltaziak M, Duraj E, Koda M, Wincewicz A, Musiatowicz M, Koda LK, Szymanska M, Lesniewicz  T, Muslatowwic  B: Expression of bcl-xl, bax, and  p53 in primary tumours and lymph node metastases in oral squamous cell  carcinoman.   Ann  NY Acad  Sci 2006;

1090: 18-25.

9-    Damron TA, Mathur S, Horton JA, Strauss J, Margulies B, Grant W, Farnum CE, Spadaro JA: Temporal changes in PTHrP, bcl-2, bax, caspase, TGF-B, and FGF-2 expression following    growth   plate   irradiation    with or without radioprotectant.  Histochem. & Cytochem. 2004; 52: 157-167.

10-  Ma  ZJ   ,   Salunga   R,  Tuggle   JT:   Gene

expression profiles of human breast cancer progression.  Gene expression profiles of human   breast   cancer   progression.   PNAS

2003; 100: 5974-5979.

11-  Mao X, Fan C, Wei J, Yao F, Jin F: Genetic mutations expression of p53 in non-invasive breast lesions.  Mol Med Report 2010; 3(6):

929-934.

12-  RajanPB, ScottDJ, PerryRH, Griffith CDM: P53 protein  expression  in ductal carcinoma in situ (DCIS) of the breast.  Br Cancer Res Treat 1997; 24: 283-290.

13-  Kayaselcuk F, Zorludemir S, Bal N, Erdogan B,  Erdogan   S,  Erman  T:  The  expression of survivin and Ki-67 in meningiomas: Correlation with grade and clinical outcome.

1Neurooncol.2004; 1&2: 209-214.

14-  Rehman  S, Crow J, Revell  PA: Bax protein expression in DCIS of the breast in relation to invasive ductal carcinoma and other molecular  markers.  Pathol Oneal Res 2000;

6: 256-263.

15- Kapucuoglu   N,   Losi   L,   Eusebi   V: Immunohistochemical localization of bcl-2 and bax proteins in situ and invasive duct breast  carcinomas.    f-lrchows  Archiv 1997;

430: 17-22.

16- Anagnostopoulos     GK,     Stefanou     D, Arkoumani   E,   Chalkley   L,   Karagiannis J, Paraskeva K, Mathou N, Dellaporta E, Tsianos E, Agnantis NJ: Expression of bax protein in gastric carcinomas. A clinico­ pathological      and     immunohistochemical study.  Acta  Gastroenterol Belg  2007;  70:

285-289.

A, Sierra  D: Bcl-xl  promotes  metastasis  of breast cancer cells by induction of cytokines resistance.  Cell Death and Differ 2000;  4:

350-359.

18- Espafia   L,  Martin  B,  Aragiies  R,  Chiva C, Oliva B, Andreu D, Sierra A: Bel-xi­ mediated changes in cetabolic pathways of breast cancer cells from survival in the blood stream  to  organ-specific  metastasis.   Am 1

Patho12005; 167: 1125-1137.

19- Korsmeyer  S: Bcl-2 gene family  and the regulation of programmed cell death. Cancer Res1999; 1:1693-1700.

20- White E: Life, death and the pursuit of apoptosis. GeneDev. 1996; 10: 1-15

21- Reed   J:   Expression    and   location   of proapoptotic   bcl-2  family   protein   bad  in human  tissues  and  tumor  cell  lines.  Am 1

Pathol1998; 152: 51--61.

22-  LU  Q,  Abel  P, Foster  C  and  LaLani  E: Bcl-2:   Role   in   epithelial    differentiation and  oncogenesis.  Human Pathol1996; 27:

102-110.

23-  Yang E, Zha J, Jockel J, Boise L, Thompson C Korsmeyer S: Bad, a heterodimeric partner for bcl-XL and bcl-2, displaces bax and promotes cell death. Cell1995; 80: 285-291.

24-  Kroemer  G: The proto-oncogene bcl-2 and its  role  in  regulating   apoptosis.   Nat  Med

1997; 3: 614--620.

25-  Oltvai ZN, Milliman CL, Korsmeyer SJ: Bcl-

2 heterodimerizes in vivo with a conserved homolog,  bax that  accelerates  programmed cell death. Cell1993; 74: 609-619.

26-  Yang E, Zha J, Jockel J, Boise L, Thompson

C Korsmeyer S: Bad, a heterodimeric partner for bcl-XL and bcl-2, displaces bax and promotes cell death. Cell1995; 80: 285-291.

27-  Zamzami   N,  Brenner   C,  Marzo  I,  Susin I, Susin S Kroemer G: Subcellular and submitochondrial   mode  of  action   of  bcl-

2  like  oncoproteins.   Oncogene 1998;   16:

2265-2282.

28-  Ioachim        EE,        Malamou-Mitsi       V, Kamina SA, Goussia AC, Agnantis NJ: Immunohistochemical  expression    of   bcl-

2   protein    in   breast    lesions:   correlation with bax, p53, Rb, C-erbB-2, EGFR and proliferation  indices.  Anticancer Res 2000;

20: 4221-4225.

29-  Skarlos  D, Gogas  H, Kiriakou  V, Margariti A, Kirkou E, Pavlaki E, Asimaki A, Toliou T, Tziortziotis D, Vamvouka C, Fountzilas G: Evaluation of the prognostic value of p53 and Bcl-2 in breast cancer patients participating