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Results for “BCL2”

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PubMed2005

Translocation t(14;18) and gain of chromosome 18/BCL2: effects on BCL2 expression and apoptosis in B-cell non-Hodgkin's lymphomas.

Gain of chromosome 18q and translocation t(14;18) are] frequently found in B-cell non-Hodgkin's lymphomas (B-NHL). Increased BCL2 transcription and BCL2 protein expression have been suggested to be the result of the gain. We utilized FISH, PCR and array CGH to study BCL2 and chromosome 18 copy number changes and rearrangements in 93 cases of B-NHL. BCL2 protein was expressed in >75% of the tumor cells in 92% of the cases by immunohistochemistry. Gain of BCL2 was associated with a 25% increase in BCL2 expression levels (immunoblotting), whereas t(14;18) resulted in a 55% increase in BCL2 levels compared to cases without BCL2 alterations. The tumor cell (spontaneous) apoptotic fractions were similar for the cases with different BCL2 genotypes. However, the normal cell apoptotic fractions were higher for the tumors with t(14;18) compared to the tumors without BCL2 alterations, while the tumors with gain of BCL2 only showed intermediate levels. Low-level gains of parts of chromosome 18 were found in 14 of the 38 B-NHL cases with t(14;18), with a consensus region 18pter-q21.33 that did not include the BCL2 gene. The 11 cases with 18q gain only showed a consensus region encompassing 18q21.2-18q21.32 and 18q21.33, which contain PMAIP1/MALT1 and BCL2, respectively.

Apoptosis↗

PubMed2000

Survival function of ERK1/2 as IL-3-activated, staurosporine-resistant Bcl2 kinases.

Bcl2 phosphorylation at Ser-70 may be required for the full and potent suppression of apoptosis in IL-3-dependent myeloid cells and can result from agonist activation of mitochondrial protein kinase C (PKC). Paradoxically, expression of exogenous Bcl2 can protect parental cells from apoptosis induced by the potent PKC inhibitor, staurosporine (stauro). High concentrations of stauro of up to 1 microM only partially inhibit IL-3-stimulated Bcl2 phosphorylation but completely block PKC-mediated Bcl2 phosphorylation in vitro. These data indicate a role for a stauro-resistant Bcl2 kinase (SRK). We show that aurintricarboxylic acid (ATA), a nonpeptide activator of cellular MEK/mitogen-activated protein kinase (MAPK) kinase, can induce Ser-70 phosphorylation of Bcl2 and support survival of cells expressing wild-type but not the phosphorylation-incompetent S70A mutant Bcl2. A role for a MEK/MAPK as a responsible SRK was implicated because the highly specific MEK/MAPK inhibitor, PD98059, also can only partially inhibit IL-3-induced Bcl2 phosphorylation, whereas the combination of PD98059 and stauro completely blocks phosphorylation and synergistically enhances apoptosis. p44MAPK/extracellular signal-regulated kinase 1 (ERK1) and p42 MAPK/ERK2 are activated by IL-3, colocalize with mitochondrial Bcl2, and can directly phosphorylate Bcl2 on Ser-70 in a stauro-resistant manner both in vitro and in vivo. These findings suggest a role for the ERK1/2 kinases as SRKs. Thus, the SRKs can serve to functionally link the IL-3-stimulated proliferative and survival signaling pathways and, in a novel capacity, may explain how Bcl2 can suppress stauro-induced apoptosis. In addition, although the mechanism of regulation of Bcl2 by phosphorylation is not yet clear, our results indicate that phosphorylation may functionally stabilize the Bcl2-Bax heterodimerization.

Animals↗

PubMed2001

Novel role for JNK as a stress-activated Bcl2 kinase.

Interleukin (IL)-3-induced Bcl2 phosphorylation at Ser(70) may be required for its full and potent antiapoptotic activity. However, in the absence of IL-3, increased expression of Bcl2 can also prolong cell survival. To determine how Bcl2 may be functionally phosphorylated following IL-3 withdrawal, a stress-activated Bcl2 kinase (SAK) was sought. Results indicate that anisomycin, a potent activator of the stress kinase JNK/SAPK, can induce Bcl2 phosphorylation at Ser(70) and that JNK1 can be latently activated following IL-3 withdrawal to mediate Bcl2 phosphorylation. JNK1 directly phosphorylates Bcl2 in vitro, co-localizes with Bcl2, and collaborates with Bcl-2 to mediate prolonged cell survival in the absence of IL-3 or following various stress applications. Dominant-negative (DN)-JNK1 can block both anisomycin and latent IL-3 withdrawal-induced Bcl2 phosphorylation (>90%) and potently enhances cell death. Furthermore, low dose okadaic acid (OA), a potent protein phosphatase 1 and 2A inhibitor, can activate the mitogen-activated protein kinases JNK1 and ERK1/2, but not p38 kinase, to induce Bcl2 phosphorylation and prolong cell survival in factor-deprived cells. Since PD98059, a specific MEK inhibitor, can only partially inhibit OA-induced Bcl2 phosphorylation but completely blocks OA-induced Bcl2 phosphorylation in cells expressing DN-JNK1, this supports the conclusion that OA may stimulate Bcl2 phosphorylation via a mechanism involving both JNK1 and ERK1/2. Collectively, these findings indicate a novel role for JNK1 as a SAK and may explain, at least in part, how functional phosphorylation of Bc12 can occur in the absence of growth factor.

Animals↗

PubMed2002

A functional role for the B56 alpha-subunit of protein phosphatase 2A in ceramide-mediated regulation of Bcl2 phosphorylation status and function.

Recently it has been shown that the potent apoptotic agent ceramide activates a mitochondrial protein phosphatase 2A (PP2A) and promotes dephosphorylation of the anti-apoptotic molecule Bcl2 (Ruvolo, P. P., Deng, X., Ito, T., Carr, B. K., and May, W. S. (1999) J. Biol. Chem. 274, 20296-20300). In cells expressing Bcl2, dephosphorylation of Bcl2 appears to be required for ceramide-induced cell death because treatment of cells with low doses of the PP2A inhibitor okadaic acid blocks Bcl2 dephosphorylation and promotes cell survival. Furthermore, the non-phosphorylatable (i.e. PP2A-resistant) gain-of-function S70E mutant Bcl2 can protect cells from ceramide-induced apoptosis. These findings support a model whereby Bcl2 function is regulated by PP2A. PP2A is a heterotrimer that contains a catalytic C-subunit, a structural A-subunit, and a regulatory B-subunit. The A- and C-subunits are fairly conserved and ubiquitously expressed, and they form the catalytic complex of the phosphatase. In contrast, there are at least three families of diverse B-subunit molecules that vary in expression temporally and by tissue type. It is hypothesized that ceramide regulates PP2A via the B-subunit. Thus, understanding the mechanism of how PP2A regulates Bcl2 phosphorylation status and how ceramide might regulate this process requires identification of the regulatory B-subunit of PP2A that comprises the Bcl2 phosphatase. Results indicate that the B56 alpha-subunit is a candidate regulatory subunit of the physiologic Bcl2 phosphatase since (a) B56 alpha associates with Bcl2 as evidenced by pull-down experiments, (b) B56 alpha co-localizes with Bcl2 in mitochondrial membranes, (c) ceramide promotes translocation of B56 alpha to mitochondrial membranes, and (d) overexpression of B56 alpha promotes mitochondrial PP2A activity and Bcl2 dephosphorylation and potentiates cell killing with ceramide. These findings suggest a role for B56 alpha in regulating the Bcl2 phosphatase.

Blotting, Western↗

PubMed1996

BCL2 oncogene protein expression in human hematopoietic precursors during fetal life.

BCL2 proto-oncogene encodes a 25-kD protein that is characteristically localized in the inner mitochondrial membrane of the cell. It has been reported that BCL2 protein has the unique functional role of blocking programmed cells death without affecting proliferation. We have analyzed the expression of the BCL2 protein in fetal hematopoietic tissues from the 10th week of gestational age onward. Fetal thymus, liver, and bone marrow and cord blood were investigated. The experiments were performed by the alkaline-antialkaline phosphatase (APAAP) technique by staining air-dried acetone-fixed cytospins and by dual-color immunofluorescent assay by staining mononuclear cell suspensions with monoclonal antibodies detecting BCL2 protein and antigens expressed by different hematopoietic subsets. Flow cytometric analyses were performed on FACSort's Comsort 32 (Becton Dickinson, San Jose, CA). The results have shown that the BCL2 protein is expressed in human fetal ontogenesis at the earliest stages examined. The major conceptual aspects of the results are 1) BCL2 is largely expressed in the hematopoietic cells during ontogenesis. BCL2+ cells include both immature and more differentiated subsets. Moreover, the 25-kD protein is expression in cell subsets well known to be high proliferating. This behavior suggests that BCL2 could have more complex functions than those previously described. 2) The expression in the major part of CD34+ cells suggests that BCL2 could play a role in stem cell survival. 3) BCL2 is expressed in not only medullary but also cortical thymocytes, where it could cooperate in positive selection processes. 4) The involvement of BCL2 in the immunosurveillance is indicated not only by its role in B and T cell lineages but also by its expression in particular subsets like that of the cytoplasmic CD3+ fetal liver NK cells. 5) The discrepancy observed between the results of transgenic mice analysis and in vitro inhibition experiments by antisense oligonucleotides performed for understanding BCL2 functions must stress the importance of the direct immunologic analysis of BCL2 in human hematopoietic cells.

Bone Marrow↗

PubMed1998

Clinical relevance of BCL2, BCL6, and MYC rearrangements in diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLCL) is characterized by a marked degree of morphologic and clinical heterogeneity. We studied 156 patients with de novo DLCL for rearrangements of the BCL2, BCL6, and MYC oncogenes by Southern blot analysis and BCL2 protein expression. We related these data to the primary site of presentation, disease stage, and other clinical risk factors. Structural alterations of BCL2, BCL6, and MYC were detected in 25 of 156, 36 of 116, and 10 of 151 patients, respectively. Three cases showed a combination of BCL2 and BCL6 rearrangements, and two cases had a combination of BCL6 and MYC rearrangements. BCL2 rearrangement was found more often in extensive (39%) and primary nodal (17%) lymphomas than in extranodal cases (4%) (P = .003). BCL2 rearrangement was present in none of 40 patients with stage I disease, but in 22% of patients with stage II to IV (P = .006). The presence of BCL2 rearrangements did not significantly affect overall survival (OS) or disease-free survival (DFS). In contrast, high BCL2 protein expression adversely affected both OS (P = .008) and DFS (P = .01). BCL2 protein expression was poorly correlated with BCL2 rearrangement: only 52% of BCL2-rearranged lymphomas and 37% of BCL2-unrearranged cases had high BCL2 protein expression. Rearrangement of BCL6 was found more often in patients with extranodal (36%) and extensive (39%) presentation versus primary nodal disease (28%). No significant correlation was found with disease stage, lymphadenopathy, or bone marrow involvement. DFS and OS were not influenced by BCL6 rearrangements. MYC rearrangements were found in 16% of primary extranodal lymphomas, versus 2% of primary nodal cases (P = .02). In particular, gastrointestinal (GI) lymphomas (5 of 18 cases, 28%) were affected by MYC rearrangements. The distinct biologic behavior of these extranodal lymphomas was reflected by a high complete remission (CR) rate: 7 of 10 patients with MYC rearrangement attained complete remission and 6 responders remained alive for more than 4 years, resulting in a trend for better DFS (P = .07). These data show the complex nature of molecular events in DLCL, which is a reflection of the morphologic and clinical heterogeneity of these lymphomas. However, thus far, these genetic rearrangements fail as prognostic markers.

Blotting, Southern↗

PubMed2006

Interphase fluorescence in situ hybridization is more sensitive than BIOMED-2 polymerase chain reaction protocol in detecting IGH-BCL2 rearrangement in both fixed and frozen lymph node with follicular lymphoma.

The detection of t(14;18)(q32;q21) is advisable for the diagnosis of follicular lymphoma (FL). In 51 patients with FL, we evaluated the applicability and sensitivity of interphase fluorescence in situ hybridization (FISH) and polymerase chain reaction (PCR) using commercially available reagents. In 23 patients, only a formalin-fixed lymph node was available. In 28 patients, both frozen and formalin-fixed lymph nodes were evaluated. Fluorescence in situ hybridization was found to be 100% applicable whatever the material type. With the use of IGH-BCL2 dual-fusion, dual-color probes, t(14;18) translocation was detected in 47 (92%) of 51 FL cases with concordant results between isolated nuclei (n = 41) and frozen cytologic imprints (n = 28). Twenty-two IGH-BCL2-positive cases were also studied on fixed sections with BCL2 split signal probes showing a BCL2 split in all. Conversely, no BCL2 split was observed in IGH-BCL2-negative cases (n = 4). Owing to DNA degradation as assessed by the failure of control genes amplification, the applicability of PCR was found to be 76% in fixed lymph nodes (n = 51). After exclusion of the 12 noninformative cases, the BIOMED-2 protocol allowed the detection of an IGH-BCL2 fusion in 25 (64%) of 39 fixed specimens with 11 PCR-negative (31%) of 36 FISH-positive cases. Even on frozen material with 100% applicability, the amplification of a BCL2-JH breakpoint was achieved in only 20 (71%) of 28 cases with 5 PCR-negative (20%) out of 25 FISH-positive cases. Therefore, FISH was found superior to PCR (using BIOMED-2 protocol) in detecting IGH-BCL2 fusion. Finally, FISH individualized 4 IGH-BCL2-negative FL cases without specific histopathologic features. With the use of split signal DNA probes, 1 case showed a trisomy of the BCL2 locus and another displayed BCL6 and IGH breakpoints that would suggest a t(3;14). Whether such IGH-BCL2-negative cases are characterized by alternative oncogenetic pathways remains to be determined.

Chromosomes, Human, Pair 14↗

PubMed2000

Synergy between Raf and BCL2 in abrogating the cytokine dependency of hematopoietic cells.

The Raf oncoprotein plays critical roles in the transmission of mitogenic signals from cytokine receptors to the nucleus. There are three Raf family members: A-Raf, B-Raf and Raf-1. Conditionally active forms of the Raf proteins were created by ligating N-terminal truncated activated forms to the estrogen-receptor (ER) hormone-binding domain resulting in beta-estradiol-inducible constructs. We introduced these chimeric deltaRaf:ER oncoproteins into the murine FDC-P1 hematopoietic cell line. Two different types of cells were recovered after drug selection in medium containing either cytokine or beta-estradiol: (1) cytokine-dependent cells that expressed the deltaRaf:ER oncoproteins; and (2) Raf-responsive cells that grew in response to the deltaRaf:ER oncoprotein. Depending upon the particular deltaRaf:ER oncoprotein, cytokine-dependent cells were recovered 10(3) to 10(5) times more frequently than Raf-responsive cells. To determine whether BCL2 could synergize with the deltaRaf:ER oncoproteins and increase the frequency of cytokine-independent cells, cytokine-dependent deltaRaf:ER-expressing cells were infected with either a BCL2 containing retrovirus or an empty retroviral vector. BCL2 overexpression, by itself, did not relieve cytokine dependency of the parental cell line. However, BCL2 overexpression increased the frequency of Raf-responsive cells approximately five- to 100-fold. Cytokine-dependent deltaRaf:ER-infected cells entered the G1 phase of the cell cycle after cytokine withdrawal and entered S phase only after cytokine addition. Raf-responsive deltaRaf:ER cells entered the G1 phase of the cell cycle after estrogen deprivation and re-entered the cell cycle after addition of either IL-3 or the estrogen receptor antagonist tamoxifen which activates the deltaRaf:ER constructs. Expression of the BCL2 oncoprotein often delayed the exit from the S and G2/M phases demonstrating the protective effects BCL2 provided to these Raf and BCL2 infected cells. The deltaRaf:ER cells expressed the deltaRaf:ER proteins and downstream MEK and ERK activities after beta-estradiol treatment. Raf-responsive cells that were also infected with BCL2 expressed higher levels of BCL2 than the cells that were not infected with BCL2. Thus BCL2 can synergize with the activated Raf in the abrogation of cytokine dependency of certain hematopoietic cells. These cells will be useful in furthering our understanding of the roles of the Raf and BCL2 oncoproteins in hematopoietic cell growth, cell cycle progression and prevention of apoptosis.

Apoptosis↗

PubMed1998

A functional role for mitochondrial protein kinase Calpha in Bcl2 phosphorylation and suppression of apoptosis.

Phosphorylation of Bcl2 at serine 70 may result from activation of a classic protein kinase C (PKC) isoform and is required for functional suppression of apoptosis by Bcl2 in murine growth factor-dependent cell lines (Ito, T., Deng, X., Carr, B., and May, W. S. (1997) J. Biol. Chem. 272, 11671-11673). Human pre-B REH cells express high levels of Bcl2 yet remain sensitive to the chemotherapeutic agents etoposide, cytosine arabinoside, and Adriamycin. In contrast, myeloid leukemia-derived HL60 cells express less than half the level of Bcl-2 but are >10-fold more resistant to apoptosis induced by these drugs. The mechanism responsible for this apparent dichotomy appears to involve a deficiency of mitochondrial PKCalpha since 1) HL60 but not REH cells contain highly phosphorylated Bcl2; 2) PKCalpha is the only classical isoform co-localized with Bcl2 in HL60 but not REH mitochondrial membranes; 3) the natural product and potent PKC activator bryostatin-1 induces mitochondrial localization of PKCalpha in association with Bcl2 phosphorylation and increased REH cell resistance to drug-induced apoptosis; 4) PKCalpha can directly phosphorylate wild-type but not phosphorylation-negative and loss of function S70A Bcl2 in vitro; 5) stable, forced expression of exogenous PKCalpha induces mitochondrial localization of PKCalpha, increased Bcl2 phosphorylation and a >10-fold increase in resistance to drug-induced cell death; and () PKCalpha-transduced cells remain highly sensitive to staurosporine, a potent PKC inhibitor. Furthermore, treatment of the PKCalpha transformants with bryostatin-1 leads to even higher levels of mitochondrial PKCalpha, Bcl2 phosphorylation, and REH cell survival following chemotherapy. While these findings strongly support a role for PKCalpha as a functional Bcl2 kinase that can enhance cell resistance to antileukemic chemotherapy, they do not exclude the possibility that another Bcl2 kinase(s) may also exist. Collectively, these findings identify a functional role for PKCalpha in Bcl2 phosphorylation and in resistance to chemotherapy and suggest a novel target for antileukemic strategies.

Antineoplastic Agents↗

PubMed1996

Clinical significance of bcl2 and p53 protein expression in diffuse large B-cell lymphoma: a population-based study.

PURPOSE: We studied the prognostic significance of bcl2 and p53 protein expression in relation to clinical and pathologic characteristics in patients with diffuse large B-cell lymphoma (LCL). PATIENTS AND METHODS: Three hundred seventy-two patients with LCL were retrieved from a population-based registry for non-Hodgkin's lymphoma (NHL). bcl2 and p53 protein expression was studied on paraffin-embedded tumor tissue by immunohistochemistry in relation to clinical factors. Response to therapy and survival were analyzed in 165 patients who were uniformly staged and treated and for whom all prognostic data were available according to the International Prognostic Index (IPI). RESULTS: Forty-five percent of tumors showed strong expression of the bcl2 protein (bcl2++), with a higher frequency in patients with primary nodal involvement. Disease-free survival (DFS) was significantly better in bcl2-negative/intermediate (bcl2-/+) cases as compared with bcl2++ cases (P = .0011). At 5 years, bcl2-/+ patients showed a DFS rate of 74%, in contrast to bcl2++ patients with a DFS rate of 41% (P = .002). Bcl2 was the strongest independent prognostic value in a multivariate analysis, with a relative risk (RR) of 3.0 in comparison to p53 expression and the clinical factors of the IPI. Overall survival (OS) was not significantly influenced by bcl2 protein expression. p53 protein expression was found in 13% of cases, with a higher frequency in patients with extensive disease. p53 expression did not influence the chance to achieve complete remission (CR) and survival. CONCLUSION: bcl2 protein is frequently expressed in LCL and is a strong independent prognostic factor for DFS. p53 expression is related with high tumor burden, but is not an independent risk factor for CR and survival.

Adult↗

PubMed2006

BCL2 expression is a prognostic marker for the activated B-cell-like type of diffuse large B-cell lymphoma.

BACKGROUND: The role of BCL2 as a predictor of survival in diffuse large B-cell lymphoma (DLBCL) is controversial. DLBCL is heterogeneous, and the expression of BCL2 is variable within the two major subgroups of DLBCL, germinal center B-cell-like (GCB) and activated B-cell-like (ABC) DLBCL, as well as primary mediastinal DLBCL. PATIENTS AND METHODS: In this study, we investigated the correlation of BCL2 expression with survival in the two major subgroups of DLBCL, as well as the mechanisms of BCL2 expression. RESULTS: There was no significant correlation between BCL2 protein expression and overall survival within the GCB subgroup, but BCL2 expression had a significant adverse effect on overall survival within the ABC subgroup (P = .008). This correlation was also observed at the mRNA level (P < .04). The difference remained significant when the analyses were performed at different cutoff values. The t(14;18) was frequently observed in the GCB subgroup and was highly associated with BCL2 expression. Patients with ABC DLBCL did not exhibit t(14;18) but had a markedly higher frequency of chromosome 18q21 amplification, on which BCL2 resides. Thus, alternative mechanisms such as 18q21 amplification or activation of the nuclear factor-kappa B pathway, as reported previously, seem to be mainly responsible for the upregulation of BCL2 expression in the ABC subgroup. CONCLUSION: Treating all DLBCL as a single entity ignores the mechanistic differences in BCL2 upregulation and obscures the prognostic significance of BCL2 expression. Hence, the significance of BCL2 and other biomarkers should be assessed in the context of DLBCL subgroups in future studies.

Aged↗

PubMed1999

BCL2 overexpression in diffuse large B-cell lymphoma.

Translocation (14;18)(q32;q21), which is detected in 20-30% of diffuse large B-cell lymphomas (DLBCL), is regarded as a major mechanism for BCL2 protein overexpression. Nevertheless, BCL2 overexpression is not always caused by t(14;18), because it is often detected in lymphomas without BCL2 rearrangement. Recent studies have shown that increased expression of BCL2 may also result from BCL2 gene amplification in DLBCL. Similarly, it has been speculated that the mutations of the open reading frame might cause increased expression of BCL2 by affecting the interactions of BCL2 with other proteins. The results obtained from studies on the association of BCL2 protein overexpression with survival of DLBCL are controversial, although a correlation with decreased overall survival seems to exist. However, BCL2 rearrangement does not seem to have any major association with poor prognosis, but it is difficult to assess its true impact on prognosis due to differences in treatment and follow-up, and methodologies applied to study the BCL2 rearrangement. This review summarizes the BCL2 expression studies in DLBCL and discusses the prognostic relevance of BCL2 overexpression and its mechanisms.

Animals↗

PubMed1999

The prognostic impact of BCL2 protein expression in acute myelogenous leukemia varies with cytogenetics.

BCL2 protein exerts an antiapoptotic effect on cells and decreases chemosensitivity. To determine whether BCL2 expression is prognostic of patient outcome in acute myelogenous leukemia (AML), we measured its level in 198 newly diagnosed, untreated AML patients by Western blotting using whole-cell lysates from low-density peripheral blood cells. BCL2 expression was not associated with the percentage of blasts (R2 = 0.10), French-American-British classification type, or cytogenetic abnormality. Smoothed martingale residual plots indicated that high BCL2 protein level was an adverse prognostic factor for patients with either favorable or intermediate prognosis cytogenetics [FIPC; inv(16), t(8:21), t(15:17), or diploid or insufficient metaphases] but was a favorable prognostic factor for patients with unfavorable prognosis cytogenetics (UC; -5, -7, +8, 11q23, Ph1, or miscellaneous changes). Patients with FIPC and high BCL2 (highest quartile) had a significantly shorter median survival (78 weeks versus not reached; P = 0.009) than did those with lower (lower three quartiles) levels of BCL2. Among those with UC, as BCL2 level decreased from the fourth quartile to the third or the combined first and second quartiles, the median survival decreased (from 94 to 45 or 17 weeks, respectively; P = 0.003). Lower expression of BCL2 in UC was associated with shorter remission duration (P = 0.05). In multivariate analyses performed using either overall or event-free survival as the end point, for either all patients or within either cytogenetic subgroup, BCL2 level was an independent prognostic factor. Similar analysis revealed that BCL2 level was an independent predictor of remission duration for UC patients as well. These data suggest that BCL2 is involved differently in different types (favorable versus unfavorable) of AML and that therapeutic strategies aimed at modulating BCL2 function may be more likely to work in patients with favorable cytogenetic abnormalities.

Adolescent↗

PubMed1996

Rearrangement of the 5' cluster region of the BCL2 gene in lymphoid neoplasm: a summary of nine cases.

Rearrangement of the BCL2 gene with the immunoglobulin (IG) genes is the most frequent genetic abnormality in B cell lymphoid neoplasms. In the majority of cases, breakages occur at two breakpoint cluster regions; major breakpoint cluster (MBR) and minor cluster region (mcr). In a minority of cases with non-Hodgkin's lymphoma (NHL) and chronic lymphocytic leukemia (CLL), rearrangements involving the 5' flanking region of the BCL2 (5'-BCL2) have been reported. Here, we investigated 196 patients with NHL and 31 with CLL, with regard to rearrangement of the BCL2 gene. Hybridization analyses using probes representing the three cluster regions revealed that a total of 57 patients had a rearrangement of the BCL2; 42 (73.7%) were within the MBR, seven (12.2%) were within the mcr, and nine (15.8%) had a rearrangement at the 5'-BCL2. The nine patients with 5'BCL2 rearrangement included two with follicular lymphoma, four with diffuse large cell lymphoma and immunoblastic variant, two with leukemic phase of follicular lymphoma, and one with CLL. Comigration analysis with probes for the IG heavy chain gene (IGH), kappa-chain gene (IG kappa) and lambda-chain gene (IG lambda), demonstrated a 5'-BCL2/IGH junction at the JH region in four patients with NHL derived from follicular center B cell. Thus, the 5'flanking region is a third cluster for recombination between the BCL2 and IGH, which is closely associated with the development of follicular center cell lymphoma. Molecular cloning of a 5'-BCL2/IGH junction demonstrated recombination of the two affected genes in divergent orientation. A 5'-BCL2/IG kappa junction was observed in two patients with immunoblastic lymphoma, and one with CLL had a 5'-BCL2/IG lambda recombination. Two patients, including one with a BCL2-MBR/JH junction, lacked obvious recombination of the 5'-BCL2 with IG genes, suggesting the presence of a deletion at the 5'-BCL2. Our findings demonstrated heterogeneity not only in clinicopathological presentation of B cell disease with rearrangement of 5'-BCL2, but also in molecular lesions resulting from the rearrangement.

Adult↗

PubMed1996

Altered methional homoeostasis is associated with decreased apoptosis in BAF3 bcl2 murine lymphoid cells.

Methional is a potent inducer of apoptosis in an interleukin 3-dependent murine lymphoid cell line BAF3 b0 when it is added to the culture medium. In these cells transfected with the bcl2 gene, BAF3 bcl2, the apoptotic-inducing activity of methional is dramatically reduced. The addition of disulfiram (an inhibitor of aldehyde dehydrogenase) in order to reduce methional oxidation brought about an increase in apoptosis in BAF3 b0 but not in BAF3 bcl2 cells. In contrast, the addition of quercetin (an inhibitor of aldehyde reductase) in an attempt to diminish methional reduction increased apoptosis in both BAF3 b0 and BAF3 bcl2 cells. The extent of DNA fragmentation in BAF3 bcl2 cells approached that in BAF3 b0 cells in the presence of quercetin and exogenous methional, suggesting a defect in methional biosynthesis in BAF3 bcl2 cells. Direct evidence for this was obtained by measuring labelled methional in cells incubated with the sodium, salt of [U-14C]4-methylthio-2-oxobutanoic acid (MTOB), the precursor of methional. The 80% decrease in labelled methional in BAF3 bcl2 compared with BAF3 b0 cells was accompanied by a concomitant rise in the transamination of [14C]MTOB to [14C]methionine in BAF3 bcl2 cells. Inhibition of the transaminase, however, by a synthetic transition-state-type compound, pyridoxal-L-methionine ethyl ester, induced apoptosis in BAF3 b0 but not in BAF3 bcl2 cells, confirming that the defect in BAF3 bcl2 cells was not in the transaminase itself but rather in the oxidative decarboxylation step MTOB --> methional. In addition, no evidence was obtained for the synthesis of [14C]malondialdehyde from [14C]methional in BAF3 bcl2 cells. As these cells show no deficiency in their content of reactive oxygen species compared with that of BAF3 b0 cells, they may possess some other defect in the beta-hydroxylase enzyme system itself.

Aldehyde Dehydrogenase↗

PubMed1991

BCL2 protein is topographically restricted in tissues characterized by apoptotic cell death.

The BCL2 protooncogene encodes an inner mitochondrial membrane protein that blocks programmed cell death. BCL2 was isolated from the chromosomal breakpoint of follicular B-cell lymphoma. Transgenic mice that overexpress BCL2 display extended survival of resting B cells. In this study we use a monospecific anti-human BCL2 antibody to define the distribution of BCL2 protein within organized tissues. BCL2 is restricted within germinal centers to the follicular mantle and to portions of the light zone implicated in the selection and maintenance of plasma cells and memory B cells. BCL2 is present in the surviving T cells in the thymic medulla. All hematopoietic lineages that derive from a renewing stem cell also display BCL2. A limited number of nonlymphoid tissues demonstrate BCL2 and can be grouped as (i) glandular epithelium in which hormones or growth factors regulate hyperplasia and involution, (ii) complex differentiating epithelium such as skin and intestine characterized by long-lived stem cells, and (iii) long-lived postmitotic cells such as neurons. Within these tissues that demonstrate apoptotic cell turnover, BCL2 is often topographically restricted to long-lived or proliferating cell zones. BCL2's function as an antidote to apoptosis may confer longevity to progenitor and effector cells in these tissues.

Adult↗

PubMed1996

BCL2 regulates neural differentiation.

A main function attributed to the BCL2 protein is its ability to confer resistance against apoptosis. In addition to the constitutively high expression of BCL2, caused by gene rearrangement in follicular lymphomas, elevated expression of the BCL2 gene has been found in differentiating hematopoietic, neural, and epithelial tissues. To address the question of whether the expression of BCL2 is a cause or consequence of cell differentiation, we used a human neural-crest-derived tumor cell line, Paju, that undergoes spontaneous neural differentiation in vitro. The Paju cell line displays moderate expression of BCL2, the level of which increases in parallel with further neural differentiation induced by treatment with phorbol 12-myristate 13-acetate. Transfection of normal human BCL2 cDNA in sense and antisense orientations had a dramatic impact on the differentiation of the Paju cells. Overexpression of BCL2 cDNA induced extensive neurite outgrowth, even in low serum concentrations, together with an increased expression of neuron-specific enolase. Paju cells expressing the anti-sense BCL2 cDNA construct, which reduced the endogenous levels of BCL2, did not undergo spontaneous neural differentiation. These cells acquired an epithelioid morphology and up-regulated the intermediate filament protein nestin, typically present in primitive neuroectodermal cells. The manipulated levels of BCL2 did not have appreciable impact on cell survival in normal culture. Our findings demonstrate that the BCL2 gene product participates in the regulation of neural differentiation.

Adolescent↗

PubMed2000

Interphase FISH detection of BCL2 rearrangement in follicular lymphoma using breakpoint-flanking probes.

Rearrangement of the BCL2 gene is an important parameter for the differential diagnosis of non-Hodgkin lymphomas. Although a relatively large proportion of breakpoints is clustered, many are missed by standard PCR. A FISH assay is therefore desired. Up to now, a lack of probes flanking the BCL2 gene has limited the possibilities for a FISH assay to an approach based on colocalization of probes for BCL2 and the immunoglobulin heavy chain (IGH) locus. Intrinsically high rates of false positive nuclei and high interobserver variability make such assays unsuitable for use on lymphoma tissue samples, where tumor cells often form only a minority of the cell population. Using YAC end cloning techniques and screening of a PAC library, we have isolated PAC clones flanking the BCL2 gene. Using these PACs, and several cosmid clones in the second BCL2 intron, we developed a segregation-based interphase FISH assay with two probe combinations enabling separate detection of 5' and 3' (mbr/mcr) breakpoints. The assay was applied to a series of 40 follicular lymphomas. To evaluate the results, the same lymphomas were analyzed by DNA fiber FISH with a 600-kb set of BCL2 DNA clones labeled in alternating colors in combination with a color barcode covering the IGH locus. This approach allowed precise mapping of BCL2 breakpoints, and simultaneously showed juxtaposition of IGH genes to BCL2. Comparison of the results of interphase and fiber FISH showed complete correlation. Five cases were negative with both FISH techniques as well as with Southern blotting. Interestingly, all of these 5 cases lacked BCL2 overexpression as determined by immunohistochemistry, against 3 of 35 rearrangement-positive follicular lymphomas. Furthermore, absence of t(14;18) seemed to be correlated with a higher histologic grade (grades 2 and 3 according to Berard). These data indicate that the segregation-based interphase FISH assay detects 100% of BCL2 rearrangements. Because interpretation of the results is straightforward and requires no extensive experience, this assay may be the best available diagnostic test for BCL2 rearrangement. Genes Chromosomes Cancer 27:85-94, 2000.

3' Untranslated Regions↗