Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death

The t(14; 18) chromosomal translocation of human follicular B-cell lymphoma juxtaposes the bcl-2 gene with the immunoglobulin heavy chain locus. The bcl-2 immunoglobulin fusion gene is markedly deregulated resulting in inappropriately elevated levels of bcl-2 RNA and protein. Transgenic mice bearing a bcl-2 immunoglobulin minigene demonstrate a polyclonal expansion of resting yet responsive IgM-IgD B cells which display prolonged cell survival but no increase in cell cycling. Moreover, deregulated bcl-2 extends the survival of certain haematopoietic cell lines following growth-factor deprivation. By using immunolocalization studies we now demonstrate that Bcl-2 is an integral inner mitochondrial membrane protein of relative molecular mass 25,000 (25k). Overexpression of Bcl-2 blocks the apoptotic death of a pro-B-lymphocyte cell line. Thus, Bcl-2 is unique among proto-oncogenes, being localized to mitochondria and interfering with programmed cell death independent of promoting cell division.     

Bcl-2 gene promotes haemopoietic cell survival and cooperates with c-myc to immortalize pre-B cells

A common feature of follicular lymphoma, the most prevalent haematological malignancy in humans, is a chromosome translocation (t(14;18] that has coupled the immunoglobulin heavy chain locus to a chromosome 18 gene denoted bcl-2. By analogy with the translocated c-myc oncogene in other B-lymphoid tumours bcl-2 is a candidate oncogene, but no biological effects of bcl-2 have yet been reported. To test whether bcl-2 influences the growth of haematopoietic cells, either alone or together with a deregulated c-myc gene, we have introduced a human bcl-2 complementary DNA using a retroviral vector into bone marrow cells from either normal or E mu-myc transgenic mice, in which B-lineage cells constitutively express the c-myc gene. Bcl-2 cooperated with c-myc to promote proliferation of B-cell precursors, some of which became tumorigenic. To determine how bcl-2 expression impinges on growth factor requirements, the gene was introduced into a lymphoid and a myeloid cell line that require interleukin 3 (IL-3). In the absence of IL-3, bcl-2 promoted the survival of the infected cells but they persisted in a G0 state, rather than proliferating. These results argue that bcl-2 provided a distinct survival signal to the cell and may contribute to neoplasia by allowing a clone to persist until other oncogenes, such as c-myc, become activated.     

Small G proteins are expressed ubiquitously in lymphoid cells and do not correspond to Bcl-2

The bcl-2 gene is consistently associated with t(14; 18) chromosomal translocations observed in a large fraction of human B-cell lymphomas. The t(14; 18) translocation results in deregulated expression of the bcl-2 gene and synthesis of inappropriately high levels of the Bcl-2 protein. Gene transfer studies suggest a role for Bcl-2 in cell survival, growth enhancement and oncogenic transformation. To test the suggestion that GTP-binding by Bcl-2 may mediate its biological effects we characterized the GTP-binding proteins in lymphoid cells expressing Bcl-2. Expression of several small GTP-binding proteins was found to be ubiquitous and did not vary with levels of Bcl-2. By using immunological, electrophoretic and cell-fractionation techniques, we separated Bcl-2 from G proteins of small relative molecular mass (Mr) and showed that it is incapable of binding GTP. Our results show that small Mr G proteins are widely expressed in lymphoid cells and that Bcl-2 is not a novel member of this GTP-binding protein family.     

Novel primitive lymphoid tumours induced in transgenic mice by cooperation between myc and bcl-2

The putative oncogene bcl-2 is juxtaposed to the immunoglobulin heavy chain (Igh) locus by the t(14;18) chromosomal translocation typical of human follicular B-cell lymphomas. The bcl-2 gene product is not altered by the translocation, but its expression is deregulated, presumably by the Igh enhancer E mu. Constitutive bcl-2 expression seems to augment cell survival, as infection with a bcl-2 retrovirus enables certain growth factor-dependent mouse cell lines to maintain viability when deprived of factor. Furthermore, high levels of the bcl-2 product can protect human B and T lymphoblasts under stress and thereby confer a growth advantage. Mice expressing a bcl-2 transgene controlled by the Igh enhancer accumulate small non-cycling B cells which survive unusually well in vitro but do not show a propensity for spontaneous tumorigenesis. In contrast, an analogous myc transgene, designed to mimic the myc-Igh translocation product typical of Burkitt's lymphoma and rodent plasmacytoma, promotes B lymphoid cell proliferation and predisposes mice to malignancy in pre-B and B lymphoid cells. Previous experiments have suggested that bcl-2 can cooperate with deregulated myc to improve in vitro growth of pre-B and B cells. Here we describe a marked synergy between bcl-2 and myc in doubly transgenic mice. E mu-bcl-2/myc mice show hyperproliferation of pre-B and B cells and develop tumours much faster than E mu-myc mice. Suprisingly, the tumours derive from a cell with the hallmarks of a primitive haemopoietic cell, perhaps a lymphoid-committed stem cell.     

The bcl-2 gene encodes a novel G protein

Little is known about the biochemical or functional nature of the proteins encoded by the bcl-2 gene, which undergoes chromosomal translocation in approximately 85% of follicular lymphoma, 20% of diffuse large cell lymphoma and 10% of chronic lymphocytic leukaemia of B cells. Translocation of bcl-2 sequences from chromosome 18 to the JH segment of the immunoglobulin gene at chromosome band 14q32 in B cells results in deregulated expression of this gene, causing high steady state levels of bcl-2 messenger RNA2. DNA sequence data indicate that bcl-2 encodes two proteins by virtue of alternative splicing, designated as Bcl-2 alpha and Bcl-2 beta, with relative molecular masses of 26,000 and 22,000 respectively. Cell fractionation experiments indicate that the bcl-2 alpha gene product is located at the inner surface of the cell membrane, suggesting a possible role in mitogenic signal transduction. We report here that Bcl-2 alpha has GTP-binding activity and a protein sequence that suggests it belongs to the small molecular weight GTP-binding protein (G protein) family.     

A non-B-DNA structure at the Bcl-2 major breakpoint region is cleaved by the RAG complex

The causes of spontaneous chromosomal translocations in somatic cells of biological organisms are largely unknown, although double-strand DNA breaks are required in all proposed mechanisms. The most common chromosomal abnormality in human cancer is the reciprocal translocation between chromosomes 14 and 18 (t(14;18)), which occurs in follicular lymphomas. The break at the immunoglobulin heavy-chain locus on chromosome 14 is an interruption of the normal V(D)J recombination process. But the breakage on chromosome 18, at the Bcl-2 gene, occurs within a confined 150-base-pair region (the major breakpoint region or Mbr) for reasons that have remained enigmatic. We have reproduced key features of the translocation process on an episome that propagates in human cells. The RAG complex--which is the normal enzyme for DNA cleavage at V, D or J segments--nicks the Bcl-2 Mbr in vitro and in vivo in a manner that reflects the pattern of the chromosomal translocations; however, the Mbr is not a V(D)J recombination signal. Rather the Bcl-2 Mbr assumes a non-B-form DNA structure within the chromosomes of human cells at 20-30% of alleles. Purified DNA assuming this structure contains stable regions of single-strandedness, which correspond well to the translocation regions in patients. Hence, a stable non-B-DNA structure in the human genome appears to be the basis for the fragility of the Bcl-2 Mbr, and the RAG complex is able to cleave this structure.     

Clonal expansion of p53 mutant cells is associated with brain tumour progression.

Tumour progression is a fundamental feature of the biology of cancer. Cancers do not arise de novo in their final form, but begin as small, indolent growths, which gradually acquire characteristics associated with malignancy. In the brain, for example, low-grade tumours (astrocytomas) evolve into faster growing, more dysplastic and invasive high-grade tumours (glioblastomas). To define the genetic events underlying brain tumour progression, we analysed the p53 gene in ten primary brain tumour pairs. Seven pairs consisted of tumours that were high grade both at presentation and recurrence (group A) and three pairs consisted of low-grade tumours that had progressed to higher grade tumours (group B). In group A pairs, four of the recurrent tumours contained a p53 gene mutation; in three of them, the same mutation was found in the primary tumour. In group B pairs, progression to high grade was associated with a p53 gene mutation. A subpopulation of cells were present in the low-grade tumours that contained the same p53 gene mutation predominant in the cells of the recurrent tumours that had progressed to glioblastoma. Thus, the histological progression of brain tumours was associated with a clonal expansion of cells that had previously acquired a mutation in the p53 gene, endowing them with a selective growth advantage. These experimental observations strongly support Nowell's clonal evolution model of tumour progression.     

Clustering of breakpoints on chromosome 11 in human B-cell neoplasms with the t(11;14) chromosome translocation

The t(11;14) (q13;q32) chromosome translocation has been reported in diffuse small and large cell lymphomas and in chronic lymphocytic leukaemia (B-CLL) and multiple myeloma. Because chromosome band 14q32 is involved in this translocation, as well as in the t(8;14) (q24;q32) translocation of the Burkitt tumour, interruption of the immunoglobulin heavy-chain locus was postulated for this rearrangement. We have cloned the chromosomal joinings between chromosomes 11 and 14 and also between chromosomes 14 and 18, in B-cell tumours carrying translocations involving these chromosomes, and suggested the existence of two translocated loci, bcl-1 and bcl-2, normally located on chromosomes 11 (band q13) and 18 (band q21) respectively, involved in the pathogenesis of human B-cell neoplasms. The results indicate that in the leukaemic cells from two different cases of CLL, the breakpoints on chromosome 11 are within 8 nucleotides of each other and on chromosome 14 involve the J4-DNA segment. Because we detected a 7mer-9mer signal-like sequence with a 12-base-long spacer on the normal chromosome 11, close to the breakpoint, we speculate that the t(11;14) chromosome translocation in CLL may be sequence specific and may involve the recombination system for immunoglobulin gene segment (V-D-J) joining.     

An inhibitor of Bcl-2 family proteins induces regression of solid tumours

Proteins in the Bcl-2 family are central regulators of programmed cell death, and members that inhibit apoptosis, such as Bcl-X(L) and Bcl-2, are overexpressed in many cancers and contribute to tumour initiation, progression and resistance to therapy. Bcl-X(L) expression correlates with chemo-resistance of tumour cell lines, and reductions in Bcl-2 increase sensitivity to anticancer drugs and enhance in vivo survival. The development of inhibitors of these proteins as potential anti-cancer therapeutics has been previously explored, but obtaining potent small-molecule inhibitors has proved difficult owing to the necessity of targeting a protein-protein interaction. Here, using nuclear magnetic resonance (NMR)-based screening, parallel synthesis and structure-based design, we have discovered ABT-737, a small-molecule inhibitor of the anti-apoptotic proteins Bcl-2, Bcl-X(L) and Bcl-w, with an affinity two to three orders of magnitude more potent than previously reported compounds. Mechanistic studies reveal that ABT-737 does not directly initiate the apoptotic process, but enhances the effects of death signals, displaying synergistic cytotoxicity with chemotherapeutics and radiation. ABT-737 exhibits single-agent-mechanism-based killing of cells from lymphoma and small-cell lung carcinoma lines, as well as primary patient-derived cells, and in animal models, ABT-737 improves survival, causes regression of established tumours, and produces cures in a high percentage of the mice.     

The t(8; 14) chromosomal translocation occurring in B-cell malignancies results from mistakes in V-D-J joining

The reciprocal chromosome translocation, t(8;14), involving the heavy chain locus on chromosome 14 and the c-myc oncogene on chromosome 8 is a characteristic of the B-cell malignancies Burkitt's lymphoma and acute lymphoblastic leukaemia (ALL). We have cloned and sequenced the t(8; 14) breakpoints of an African Burkitt's lymphoma cell line, P3HR-1, and a pre-B cell ALL cell line, 380. In each case the region of chromosome 8 involved has recombined with a JH region on chromosome 14. The two sites of breakage on chromosome 8 lie within 70 base pairs (bp) of one another. At each joining site, sequences homologous to the signal sequences thought to be recognized by the V-D-J recombinase were identified, as were N regions. In B-cell chronic lymphocytic leukaemias (B-CLL) carrying the t(11; 14) chromosome translocation and in follicular lymphomas carrying the t(14; 18) translocation, the V-D-J recombinase is implicated in the mechanism of chromosomal translocations. We speculate that the same enzymatic mechanism is responsible for the t(8; 14) translocations in African Burkitt's lymphoma and pre-B cell ALL.     

Breakpoints in the human T-cell antigen receptor alpha-chain locus in two T-cell leukaemia patients with chromosomal translocations

Specific chromosomal translocations have been observed in several human and animal tumours and are believed to be important in tumorigenesis. In many of these translocations the breakpoints lie near cellular homologues of transforming genes, suggesting that tumour development is partly due to the activation of these genes. The best-characterized example of such a translocation occurs in mouse plasmacytoma and human B-cell lymphoma, where c-myc, the cellular homologue of the viral oncogene myc, is brought into close proximity with either the light- or heavy-chain genes of the immunoglobulin loci, resulting in a change in the regulation of the myc gene. T-cell malignancies also have characteristic chromosomal abnormalities, many of which seem to involve the 14q11-14q13 region. This region has recently been found to contain the alpha-chain genes of the human T-cell antigen receptor. Here we determine more precisely the chromosome breakpoints in two patients whose leukaemic T cells contain reciprocal translocations between 11p13 and 14q13. Segregation analysis of somatic cell hybrids demonstrates that in both patients the breakpoints occur between the variable (V) and constant (C) region genes of the T-cell receptor alpha-chain locus, resulting in the translocation of the C-region gene from chromosome 14 to chromosome 11. As the 11p13 locus has been implicated in the development of Wilms' tumour, it is possible that either the Wilms' tumour gene or a yet unidentified gene in this region is involved in tumorigenesis and is altered as a result of its translocation into the T-cell receptor alpha-chain locus.     

Correlation between immunoglobulin light chain expression and variant translocation in Burkitt's lymphoma

Burkitt's-type lymphomas-leukaemias (BL) are monoclonal proliferations of malignant B lymphocytes. Irrespective of whether they carry the Epstein-Barr virus (EBV) genome, these tumour cells have been shown consistently to have one of the specific reciprocal chromosome translocations, t(8; 14), t(2; 8) or t(8; 22), involving the long arm of chromosome 8 (on 8q24) and chromosome 14, 2 or 22 (on 14q32, 2p12 and 22q11, respectively). The latter chromosomes have been shown recently to carry genes for immunoglobulin (Ig) heavy chains, and kappa and lambda light chains, respectively. Furthermore, the localization of kappa light chains within 2pcen-2p13 encompasses the breakpoint observed in Burkitt's translocation (2p12). It was therefore considered of interest to determine whether the expression of immunoglobulin chains in BL cells is related to the type of chromosomal anomalies observed. We report here that there is a direct relationship between expression of immunoglobulin light chains and specific type of translocation: BL cells with t(8; 22) express lambda chains, whereas those with t(2; 8) express kappa chains.     

Peripheral neuropathies, hepatocellular carcinomas and islet cell adenomas in transgenic mice

The ability to introduce foreign DNA into the genome of mice offers unique opportunities to produce new models of disease process. Recent experiments have shown that integration and expression of simian virus 40 (SV40) T antigen genes and the murine mammary tumour virus (MMTV)-myc genes in transgenic mice can lead to the development of neoplasia in a remarkably tissue-specific manner. In the case of SV40-bearing mice, tumours consistently develop in the choroid plexus. In the accompanying paper, we show that the 72-base pair (bp) enhancer in the SV40 genome is instrumental in directing tumorigenesis to the choroid plexus. However, when the enhancer is deleted from a construction also containing the metallothionein-human growth hormone fusion gene (SV delta e-MGH), an entirely new pattern of pathology results. The present report focuses on transgenic mice carrying this construct; they develop demyelinating peripheral neuropathies, hepatocellular carcinomas and islet cell adenomas.     

Cooperative interaction between c-myc and bcl-2 proto-oncogenes.

The bcl-2 proto-oncogene is activated by translocation in a variety of B-lymphoid tumours and synergizes with the c-myc oncogene in tumour progression. The mechanism of synergy is unclear but bcl-2 expression inhibits apoptosis, a property presumably pertinent to its proto-oncogenic mode of action. We have shown that the c-myc gene is a potent inducer of apoptosis, in addition to its established role in mitogenesis. Here we show that expression of the bcl-2 protein, Bcl-2, specifically abrogates c-myc-induced apoptosis without affecting the c-myc mitogenic function. This provides a novel mechanism for oncogene cooperation, of potential importance both in carcinogenesis and in the evolution of drug resistance in tumours.     

小鼠视神经再生研究动物模型的建立

目的总结制作小鼠视神经完全截断性动物模型作为视神经再生研究的经验和体会。方法将雄性Bcl-2高表达转基因小鼠(Bcl-2 transgenic mice)和受GFAP启动子控制表达疱疹病毒-胸苷激酶转基因雌性小鼠(GFAP-TK)交配产生的4只8~12周成年小鼠(20~30g),Bcl-2/GFAP-TK双转基因小鼠作为实验组,同周龄4只Bcl-2转基因小鼠作为对照组。其中Bcl-2/GFAP-TK双转基因小鼠皮下植入缓释泵,连续7d释放更昔洛韦(GCV)100mg.kg-1.d-1以选择性地去除视神经损伤后激活的星形胶质细胞。更昔洛韦缓释泵植入术后2d在两组动物中制作右侧单眼标准完全性视神经钳夹损伤模型,视神经钳夹10d后获取组织标本。采用免疫荧光染色特异性检测再生轴突纤维并进行定量分析;结合罗丹明的霍乱毒素B亚单位(CTB-R)或增强表达绿色荧光蛋白的复制缺陷型腺相关病毒(AAV-EGFP)用作顺行性标记物以显示再生轴突是否到达大脑靶器官。结果在Bcl-2/GFAP-TK双转基因小鼠中存在免疫荧光阳性的再生视神经轴突,再生轴突计数为71.99±24.04,并可见生长锥(growth cone)样结构,但是再生轴突纤维未能延伸达到大脑靶器官。在对照组Bcl-2转基因小鼠中未见明显再生迹象。结论小鼠视神经完全截断性动物模型可用于视神经病变的再生研究。     

新近发现的恶性肿瘤的染色体异常

作者报告一组新近发现的恶性肿瘤的染色体异常,均属少见的染色体变异.系采用淋巴结或骨髓直接法制备染色体标本.恶性淋巴瘤核型为14q~+t(2;14);慢性粒细胞白血病核型为pht(2;22).讨论了癌基因与染色体易位的关系.