Bcl-2

Oncogene B-Cell leukemia 2

Gene: maps to 18q21.3 (Tsujimoto Y. et al., 1984).
mRNA: the three bcl-2 transcripts, which are 8.5, 5.5, and 3.5 kilobases (kb) long, overlap within the first exon, but only the 8.5-kb and 5.5-kb transcripts contain sequences of the second exon. The 8.5-kb and 5.5-kb transcripts seem to use different polyadenylylation sites (Tsujimoto Y. and Croce C.M., 1986).
Protein: sequence analysis of the cDNA clones corresponding to the 5.5-kb and 3.5-kb mRNAs indicates that the two bcl-2 transcripts carry two overlapping open reading frames, one of which is 717 nucleotides long and codes for a protein (bcl-2 alpha) of 239 amino acids and a molecular mass of 26 kDa, while the other codes for a protein of 205 amino acids (bcl-2 beta, molecular mass 22 kDa) that is identical to bcl-2 alpha except at the hydrophobic carboxyl terminus (Tsujimoto Y. and Croce C.M., 1986).

The bcl-2 gene product (Bcl-2) is involved in the control of programmed cell death, acting as a cell-death suppressor. A group of genes with sequence homology to Bcl2 has been described. The most distinctive feature of this gene family is that their expression modulates cell death (Oltvai Z.N. and Korsmeyer S.J. , 1994). Genes of this family can be divided into 2 functionally antagonistic groups: (i) cell-death suppressors, such as bcl2, and (ii) cell-death promoters, such as bax. Their protein products can homo- or heterodimerise, and regulation of cell death may be achieved through competitive dimerisation. These proteins are expressed differentially among different cell types and stages of differentiation; and their biological effects depend on their level, selective expression and dimerisation status (Oltvai Z.N. and Korsmeyer S.J. , 1994). The relative expression of the different bcl-2 family genes is controlled by several factors, including p53, which may render the cells more susceptible to apoptosis by directly lowering the Bcl2 to Bax ratio (Miyashita T. and Reed J.C., 1995; Veronese S. et al., 1998).
X-ray diffraction and nuclear magnetic resonance studies have indicated that the structure of the family of Bcl-2 proteins consists of two central, primarily hydrophobic alpha-helices, surrounded by amphipathic helices, and that a 60-residue loop connects the two-helices. This loop is flexible, corresponds to a region of low sequence homology and variable size among Bcl-2 family members, and is not essential for apoptotic activity (Muchmore S.W. et al., 1996).

Cell lines:
- In a series of breast cancer cell (BCC) lines, an inverse correlation was found between the expression of Bcl-2 and p53. BT20, Hs578T, MDA-MB-468, and SK-BR-3 BCC expressed high levels of p53 and no Bcl-2; MDA-MB-436, -157, -361, and MCF-7 BCC expressed Bcl-2 and very little, if any, p53; finally, BT474, HBL-100, MDA-MB-231 and -435S BCC expressed comparable levels of the two proteins. Overexpression of a mutant p53 in MCF-7 BCC could induce down-regulation of Bcl-2 both at protein and mRNA level. However, the promoter region of the human bcl-2 gene does not contain the negative regulatory elementresponsible for the down-regulation (Haldar S. et al., 1994).

- The level of Bcl-2 expression was found to be higher in MCF-7 than in MDA-MB-231 BCC line (Kandouz M. et al., 1996).

- Several groups have reported an up-regulation of Bcl-2 mRNA and protein by estradiol in MCF-7 BCC. This effect was reversed by the antiestrogens tamoxifen, 4-hydroxytamoxifen, and RU58668. On the other hand, in T-47D BCC, the synthetic progestin ORG 2058 was able to reduce the level of Bcl-2, an effect reversed by the anti-progestin RU486 (Kandouz M. et al., 1996; Leung L.K. and Wang T.T.Y., 1999).

- Taxotere, a drug affecting the integrity of microtubules was shown to induce Bcl-2 phosphorylation in various cancer cell lines, including MCF-7 BCC. This effect was also observed with other anticancer drugs (taxol, vincristin, vinblastine,...) acting on microtubules, but not with drugs damaging DNA (5'-fluorouracil, methotrexate, cisplatin, doxorubicin,...). Phosphorylation of Bcl-2 occurred in G₂-M, the phase of the cell cycle in which this class of microtubules-affecting drugs is active (Haldar S. et al., 1997).

- In estrogen receptor-positive MCF-7 BCC, tamoxifen (TAM) was shown to induce time- and concentration-dependent down-regulation of Bcl-2 at both the mRNA and protein level. Down-regulation of Bcl-2 correlated with TAM-induced apoptosis. In addition, estradiol treatment significantly increased Bcl-2 protein expression and blocked the reduction of Bcl-2 by TAM. TAM did not, however, affect Bax, Bcl-X(L), or p53 expression at the mRNA or protein level (Zhang G.J. et al., 1999).

- Whether bcl-2 could be involved in the modulation of the angiogenic phenotype of MCF-7/Adr (adriamycin-resistant) BCC was examined. Four bcl-2-overexpressing clones, a control transfectant clone, and the MCF-7/Adr parental line were used for in vitro and in vivo experiments. bcl-2 overexpression enhanced the synthesis of the hypoxia-stimulated VEGF protein and mRNA. Northern blot analysis demonstrated an increased VEGF mRNA expression in bcl-2-overexpressing clones, and reverse transcription-polymerase chain reaction showed higher levels of the VEGF(121) and VEGF(165) mRNA isoforms, which are the most active in eliciting angiogenesis. When incorporated into matrigel, supernatants of bcl-2-transfected cells cultured under hypoxic conditions induced an increased angiogenic response in C57BL/6 mice compared with that of control clone. Tumors from bcl-2 transfectants demonstrated increased VEGF expression and neovascularization as compared to the parental line, whereas the apoptosis in in vivo xenografts was similar in control and bcl-2 transfectants. The effect of bcl-2 on angiogenesis was not mediated by p53 protein (Biroccio A. et al., 2000).

- Bcl-2-mediated regulation of NF-kappaB-transcription-factor activity may lead to the up-regulation of MMP9 transcription in MCF-7/Adr BCC (Ricca A. et al., 2000).

Tumors:
- In a study of 283 node-negative breast cancers, a significantly higher fraction of Bcl-2-positive cells was observed in small, ER-positive, slowly proliferating ([³H]thymidine-labeling index), and p53-negative tumors than in large, ER-negative, rapidly proliferating, and p53-positive tumors. the predictive role of Bcl-2 expression on 6-year relapse-free and overall survival was found to be mainly dependent on p53 expression (Silvestrini R. et al., 1994).

- By Northern-blotting, Bcl-2 mRNA was detected in all breast cancer cell lines (MCF-7, T-47D, CAL51, R30C), normal breast-tissue samples (n=3), and breast tumors (n=10) analysed. No major quantitative difference was seen between these cell lines and tissue samples (Bargou R.C. et al., 1995).

- In a series of 239 breast carcinomas, high Bax expression was associated with high Bcl2 expression (Veronese S. et al., 1998).

- Bcl-2 was found to be expressed in 99 of 130 fine-needle aspirates from patients with primary breast carcinoma. Bcl-2 was correlated with positive estrogen receptor (P<0.001) and progesterone receptor (P<0.001) status and inversely correlated with p53 (P=0.0036), Ki-67 (P=0.0073), and nuclear cytologic grade (P<0.001) (Bozzetti C. et al., 1999).

Bargou R.C. et al. (1995) Expression of the bcl-2 gene family in normal and malignant breast tissue: low bax-alpha expression in tumor cells correlates with resistance towards apoptosis. Int J. Cancer 60, 854-859.
Biroccio A. et al. (2000) Bcl-2 overexpression and hypoxia synergistically act to modulate vascular endothelial growth factor expression and in vivo angiogenesis in a breast carcinoma line. FASEB J. 14, 652-660.
Bozzetti C. et al. (1999) Bcl-2 expression on fine-needle aspirates from primary breast carcinoma. Cancer 87, 224-230.
Cleary M.L. et al. (1986) Cloning and structural analysis of cDNAs for bcl-2 and a hybrid bcl-2/immunoglobulin transcript resulting from the t(14;18) translocation. Cell 47, 19-28.
Haldar S. et al. (1994) Down-regulation of bcl-2 by p53 in breast cancer cells. Cancer Res. 54, 2095-2097.
Haldar S. et al. (1997) Bcl2 is the guardian of microtubule integrity. Cancer Res. 57, 229-233.
Kandouz M. et al. (1996) Antagonism between estradiol and progestin on Bcl-2 expression in breast-cancer cells. Int. J. Cancer 68, 120-125.
Leung L.K. and Wang T.T.Y. (1999) Paradoxical regulation of Bcl-2 family proteins by 17beta-oestradiol in human breast cancer cells MCF-7. Br. J. Cancer 81, 387-392.
Miyashita T. and Reed J.C. (1995) Tumor suppressor p53 is a direct transcriptional activator of the human bax gene. Cell 80, 293-299.
Muchmore S.W. et al. (1996) X-ray and NMR structure of human Bcl-X_L, an inhibitor of programmed cell death. Nature 381, 335-341.
Oltvai Z.N. and Korsmeyer S.J. (1994) Checkpoints of dueling dimers foil death wishes. Cell 79, 189-192.
Ricca A. et al. (2000) bcl-2 over-expression enhances NF-kappaB activity and induces mmp-9 transcription in human MCF7(ADR) breast-cancer cells. Int. J. Cancer 86, 188-196.
Silvestrini R. et al. (1994) The Bcl-2 protein: a prognostic indicator strongly related to p53 protein in lymph-node negative breast cancer patients. J. Natl. Cancer Inst. 86, 499-504.
Teixeira C. et al. (1995) Estrogen promotes chemotherapeutic drug resistance by a mechanism involving bcl-2 protooncogene expression in human breast-cancer cells. Cancer Res. 55, 3902-3907.
Tsujimoto Y et al. (1984) Cloning of the chromosome breakpoint of neoplastic B cells with the t(14;18) chromosome translocation. Science 226, 1097-1099.
Tsujimoto Y. and Croce C.M. (1986) Analysis of the structure, transcripts, and protein products of bcl-2, the gene involved in human follicular lymphoma. Proc. Nat. Acad. Sci. 83, 5214-5218.
Veronese S. et al. (1998) Bax immunohistochemical expression in breast carcinoma: a study with long term follow-up. Int. J. Cancer 79, 13-18.
Zhang G.J. et al. (1999) Tamoxifen-induced apoptosis in breast cancer cells relates to down-regulation of bcl-2, but not bax and bcl-X(L), without alteration of p53 protein levels. Clin. Cancer Res. 5, 2971-2977.

Bax, BAG-1