High Levels Of Bcl-2 Expression Research Articles

Pro- and Antiapoptotic Proteins Expression on Bone Marrow and Peripheral Blood CD34+Cells in Acute Leukemia (AL) Patients

Abstract 4996It was shown that drug resistance, poor-risk cytogenetics and poor prognosis in AL is associated with high level of Bcl-2 expression and low Bax/Bcl-2 ratio (<0,3). Fas-antigen (CD95) as a protein triggering the extrinsic apoptotic pathway is differently expressed on hematopoietic precursors. More immature CD34+/CD38- AML blast cells have lower expression of Fas/Fas-L and lower Fas-induced apoptosis than CD34+/CD38+cells. CD34+/CD38− leukemia precursors also have a reduced sensitivity to daunorubicin in vitro and increased expression of multidrug resistance genes (mrp/lrp). CD34+ leukemia cells have not yet been properly characterized regarding the expression of angiotensin converting enzyme (ACE) which regulatory influence on hematopoiesis is now beeing extensively investigated. ACE expression on blast cells is high, but it’s still unknown how CD34+ACE+ leukemia cells behave after chemotherapy. Recent publications indicate that CD34+ACE+ hematopoietic precursors transplanted into NOD/SCID mice contribute 10-fold higher numbers of multilineage blood cells than their CD34+ACE- counterparts.We have studied the dynamics of Bcl-2, Bax, CD95 and ACE expression on CD34+ cells in peripheral blood (PB) and bone marrow (BM) in AL pts during treatment. PB and BM samples were collected before and on +36 day after chemotherapy. The antigens were detected by flow cytometry using monoclonal antibodies. We calculated 10 000 cells in each sample.19 pts were included in the study: 10 - AML and 9 - ALL. The control group comprised 8 healthy donors. At time of diagnosis there were 40±5,7% of CD34+ cells in BM and 26±4,9% - in PB. There was no significant difference between AML and ALL. CD34+ cells in BM and PB of healthy donors constituted 1,6% and 0,27%, respectively. After induction therapy (+36 day) CD34+ cells decreased in BM to 6,1%±3,3 (p=0,0001), in PB to 3,7%± 2,7 (p=0,0008) in all pts. The data on antigens expression on CD34+ cells of BM and PB are presented in table 1CD34+/Bcl-2+CD34+/Bax+CD34+/CD95+CD34+/ACE+BMPBBMPBBMPBBMPBAML pts n=100 day38±11,6*41±1424,4±7,929,2±7,6*16,4±8,523,2±7,821,7±9,520,8±8,7*36 day13,5±3,4**23,7±5**46,2±11,550,3±1119,9±5,536,4±1034±6,635±9,2**ALL pts n=90 day36±1133,7±1246,2±9,437,4±3,7*3,4±1,1*7,1±2,5*41±10,933,2±9,7*36 day18,4±5,826±8,938±11,840,5±1026,2±9,1**40,9±9,2**34±1062,8±10**Donors n=811,7±1,626,1±5,922,8±467,8±6,713,4±3,247,7±11,628±5,368,2±10,2*− p<0.05 compare with donors**− p<0.05 compare with day 0CD34/Bcl-2 expression in BM in AML pts is significantly higher (p=0,04) at the diagnosis comparing with donors. CD34/Bcl-2 expression in PB in AML pts and in BM and PB in ALL pts is higher too, but not significantly. This expression level decreased substantially in BM and PB in AML pts on +36 day comparing with day 0 (p<0,05). We did not found significant changes in ALL pts. CD34/Bax expression in PB is significantly lower (p=0,003) both in AML and ALL pts in comparison with donors. In AML, not in ALL, chemotherapy caused augmentation of Bax expression in CD34+ BM and PB cells on +36 day.BM and PB CD34+ cells in donors had different expression characteristics of Bcl-2 and Bax, demonstrating much higher level of pro- and antiapoptotic markers in PB cells. On the contrast CD34+ leukemia cells in BM and PB had similar characteristics regarding CD34/Bcl-2 and CD34/Bax expression. This fact demonstrates the heterogeneity of donor CD34+cells in BM and PB and points that leukemia CD34+cells in BM and PB are rather similar.CD95 expression on CD34+ BM and PB before treatment is significantly lower (p=0,01, p=0,008) in ALL pts in comparison with donors, and this expression level increased after chemotherapy (p<0,05). CD34/CD95 expression in AML pts is similar with donors, and we didn’t find changes after treatment.CD34/ACE coexpression in BM cells of leukemia pts and donors did not differ much at any time of evaluation. But CD34/ACE expression in PB cells of AML and ALL pts was much lower (p<0,05) than in donors and substantially increased on the day 36.So, our data demonstrate that Bcl-2, Bax, CD95 and ACE expression on CD34+ cells in AL pts and donors significantly differs. The chemotherapy provokes critical changes in CD34/CD95 expression in BM and PB in ALL pts, CD34/Bcl-2 expression in AML pts and ÑÂ34/ACE expression in PB in all AL pts. Disclosures:No relevant conflicts of interest to declare.

Apoptosis: A barrier against cancer no more?

Apoptosis: A barrier against cancer no more?

Abstract 3362: IL-6 promotes head and neck tumor metastasis by inducing epithelial-mesenchymal transition via the JAK-STAT3-SNAIL signaling pathway

Abstract Distant metastases in head and neck cancer patients almost invariably herald a poor prognosis. Five year survival rates for early stage localized head and neck cancers are over 80% but this drop to 40% where disease has spread to neck nodes, and to below 20% for patients with distant metastatic disease. Uncontrolled cell proliferation and angiogenesis are key characteristics of initiation and early growth of epithelial origin cancers. However, it is the subsequent acquisition of motility and invasiveness that leads to metastatic dissemination of these tumor cells. A key process in this metastatic cascade that converts an adherent epithelial cell into a migratory cell which can invade through the extracellular matrix is known as epithelial-mesenchymal transition (EMT). EMT plays a critical role in epithelial tumor cell metastasis and is commonly observed in tumor samples from head and neck cancer patients. However, the precise molecular events that initiate this complex process of EMT in head and neck cancers are poorly understood. There is increasing evidence that suggest an important role of tumor microenvironment in promoting EMT changes in tumor cells. We have previously shown that head and neck tumors exhibit significantly higher levels of Bcl-2 expression in tumor associated endothelial cells and overexpression of Bcl-2 alone in tumor-associated endothelial cells was sufficient to enhance tumor metastasis of oral squamous cell carcinoma in a SCID mouse model. In this study, we show that endothelial cells expressing Bcl-2 (EC-Bcl-2) when co-cultured with head and neck tumor cells significantly enhance EMT-related changes in tumor cells and this EC-Bcl-2-induced EMT in tumor cells is mediated predominantly by IL-6 secretion. Treatment with recombinant IL-6 or stable IL-6 overexpression in CAL27 cells or normal oral epithelial cells (NOE) significantly induced the expression of vimentin while repressing E-cadherin expression via the JAK/STAT3/Snail signaling pathway. This phenotype was further associated with enhanced tumor and NOE cell scattering and motility. STAT3 knock-down significantly reversed IL-6-mediated tumor and NOE cell motility by inhibiting FAK activation. Furthermore, tumor cells overexpressing IL-6 showed marked increase in lymph node and lung metastasis, in vivo. These results suggest a novel function for IL-6 in mediating head and neck tumor EMT and metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3362. doi:10.1158/1538-7445.AM2011-3362

The expression of Bcl-2 anti- apoptotic protein in colorectal adenocarcinoma

&amp;lt;p&amp;gt;&amp;lt;strong&amp;gt;Introduction. &amp;lt;/strong&amp;gt;The Bcl-2 gene codes an oncoprotein, inhibits a programmed cell death or apoptosis and it plays a very important role in colorectal cancerogenesis. The aim of our study is to determine Bcl-2 expression in colorectal carcinomas in relation with the stage of disease, histology of tumor type, localization and macroscopic growth pattern. &amp;lt;strong&amp;gt;Methods.&amp;lt;/strong&amp;gt; Immunohistochemichal detection of Bcl-2 protein expression was carried out on 90 resected colorectal carcinomas. The patients were divided into three groups according to the degree of Bcl-2 expression in a tumor: group 0 - there were no cells with positive immunohistochemical reaction; group I- up to 20 % of positive cells, and group II- above 20% of positive cells. The groups were compared in relation to the stage of disease, T stage of local spread of disease, histology of tumor type, localization and macroscopic growth pattern. &amp;lt;strong&amp;gt;Results.&amp;lt;/strong&amp;gt; 58% of patients at the second stage of disease had no expression of Bcl-2. Higher percentage (61%) of the patients with metastases ( stages III and IV) had high level of Bcl-2 expression. Tumors with polipoid growth pattern have higher level of Bcl-2 expression. &amp;lt;strong&amp;gt;Conclusion. &amp;lt;/strong&amp;gt;There is a statisticaly significant difference in Bcl-2 protein expression in patients surgically treated at the II and III stage of disease, stage T3a/b, T3c/d of local spread of disease and tumors whith polipoid growth pattern in relation to infiltrative growth pattern.&amp;lt;/p&amp;gt;

Association of expression of bcl-2 with outcome in non-small cell lung cancer

e22039 Background: BCL-2 promotes cell survival by inhibiting adapters needed for the activation and cleavage of caspases thus blocking the proteolytic cascade that ultimately dismantles the cell. It is preferentially expressed in squamous cell carcinomas of the lung and has been investigated as a potential prognostic parameter in lung cancer patients with conflicting results. Here, we quantitatively assessed BCL-2 protein expression in two large and independent data sets to investigate the impact of BCL-2 on patient survival. Methods: AQUA, a fluorescent-based method for analysis of in situ protein expression, was used to measure BCL-2 protein levels and classify tumor by BCL-2 expression in a cohort of 180 lung cancer patients from Yale New Haven Hospital (training set). An independent cohort of 360 lung cancer patients from Sotiria General Hospital and Patras University Hospital in Greece was used to validate BCL-2 classification and evaluate outcome (validation set). Results: Tumors expressed BCL-2 in 57% and 53% of the cases in training and validation cohorts respectively and squamous cell carcinomas expressed higher levels of BCL-2 expression compared to adenocarcinomas (mean AQUA score 42 and 26 respectively, p=0.007); BCL-2 was not associated with other standard clinical or pathological characteristics. Survival analysis showed that patients with high BCL-2 expression had a longer median overall survival compared to the low expressers (20 vs 15 months, log rank p=0.016). Multivariate analysis revealed an independent lower risk of death for lung cancer patients with BCL-2 expressing tumors (HR=0.58, 95% CI 0.39–0.86, p=0.006). Conclusions: BCL-2 expression defines a subgroup of patients with a favorable outcome and may be useful for prognostic stratification of lung cancer patients as well as incorporation of BCL-2 into clinical decisions. [Table: see text]

CD34+ leukemic subpopulation predominantly displays lower spontaneous apoptosis and has higher expression levels of Bcl-2 and MDR1 genes than CD34− cells in childhood AML

In view of obscure clinical and biological significance of leukemic cells heterogeneity, we studied the efficacy of apoptosis, proliferation, and expression levels of the Bcl-2, MDR1, LRP, and BCRP genes in sorted CD34+ and CD34- subpopulations of childhood AML leukemic samples. In five out of nine cases, CD34+ cells were less sensitive to spontaneous apoptosis and had from 1.2- to 5.0-fold higher expression levels of Bcl-2 (eight of ten) and from 1.5- to 28.7-fold higher expression levels of MDR1 (eight of ten). The expression levels of the LRP gene were from 1.1- to 1.8-fold higher in CD34+ subpopulations (five of ten cases), and the expression levels of the BCRP gene were from 1.1- to 22.4-fold higher in CD34+ leukemic cells (six of ten). In all M4 cases, the expression levels of LRP were higher in the CD34- subpopulation. Significant differences in the patterns of genes expression between patients do not allow us to conclude that the CD34+ fractions have more resistant phenotype than the CD34- subpopulations. Nevertheless, distinctions between CD34+ and CD34- cells may lead to different chemosensitivities between leukemic subpopulations in vivo and may determine the alteration of the leukemic immunophenotype during treatment and in relapse.

The cellular concentration of Bcl-2 determines its pro- or anti-apoptotic effect

Bcl-2 is an oncoprotein that is widely known to promote cell survival by inhibiting apoptosis. We explored the consequences of different expression paradigms on the cellular action of Bcl-2. Using either transient or stable transfection combined with doxycycline-inducible expression, we titrated the cellular concentration of Bcl-2. With each expression paradigm Bcl-2 was correctly targeted to the endoplasmic reticulum and mitochondria. However, with protocols that generated the greatest cellular concentrations of Bcl-2 the structure of these organelles was dramatically altered. The endoplasmic reticulum appeared to be substantially fragmented, whilst mitochondria coalesced into dense perinuclear structures. Under these conditions of high Bcl-2 expression, cells were not protected from pro-apoptotic stimuli. Rather Bcl-2 itself caused a significant amount of spontaneous cell death, and sensitised the cells to apoptotic agents such as staurosporine or ceramide. We observed a direct correlation between Bcl-2 concentration and spontaneous apoptosis. Expression of calbindin, a calcium buffering protein, or an enzyme that inhibited inositol 1,4,5-trisphosphate-mediated calcium release, significantly reduced cell death caused by Bcl-2 expression. We further observed that high levels of Bcl-2 expression caused lipid peroxidation and that the deleterious effects of Bcl-2 could be abrogated by the reactive oxygen species (ROS) scavenger Trolox. When stably expressed at low levels, Bcl-2 did not corrupt organelle structure or trigger spontaneous apoptosis. Rather, it protected cells from pro-apoptotic stimuli. These data reveal that high cellular concentrations of Bcl-2 lead to a calcium- and ROS-dependent induction of death. Selection of the appropriate expression paradigm is therefore crucial when investigating the biological role of Bcl-2.

Exploitation of drug-induced Bcl-2 overexpression for restoring normal apoptosis function: A promising new approach to the treatment of multidrug resistant cancer

Agents antagonizing the anti-apoptotic Bcl-2 protein have been shown to restore normal apoptotic processes in cancer cells. Since upregulation of Bcl-2 is often observed in recurrent or refractory hematological malignancies, we were prompted to investigate whether drug-selected leukemia cells overexpressing Bcl-2 were more susceptible to Bcl-2 antagonists. The current study showed that a camptothecin (CPT)-selected human leukemia cell line (CPT-K5) had remarkably higher expression levels of Bcl-2 than its drug sensitive parental cell (RPMI 8402). A small molecule inhibitor of Bcl-2, HA14-1, induced much more extensive apoptosis in CPT-K5 than in RPMI 8402 cells, as characterized by DNA fragmentation, loss of mitochondrial membrane potential (Δ ψ m) and plasma membrane integrity, as well as the activation of caspase. Taken together, these findings suggest that small molecule Bcl-2 inhibitors may represent a promising class of alternative agents for the treatment of Bcl-2 overexpressed refractory or recurrent hematological malignancies when conventional chemotherapy fails.

Oblimersen

Oblimersen

Bcl2 Expression Predicts Better Overall Survival in 90 Patients with Mantle Cell Lymphoma.

The expression of bcl2 was typically observed in follicular lymphoma with a t(14;18) but also in CLL and mantle cell lymphoma (MCL). Up to now no study investigated the expression of bcl2 in correlation with the clinical outcome in a large collective of patients with MCL. We investigated immunohistochemically the expression of the apoptotic marker bcl2 in relation to the clinical course. 90 biopsy specimens were investigated immunohistochemically with antibodies against CD20, CD5, CD3, CD23, cyclin D1 and bcl2. The expression was analyzed in three groups: negative, 5–50% positive cells (weak positive) and more than 50% positive cells (strong positive). The expression profils were compared with the overall survival data analysed according to Kaplan and Meier. In none of thae cases a negative staining was found. 16 cases (18%) had a weak and 74 cases (82%) a strong bcl2 expression. Patients with mantle cell lymphoma that had a weak positive expression (n=16) had a median overall survival time of 21 months compared to 32 months for patients with a strong expression (n=74) (p=0.0402). In contrast to DLBL high levels of bcl2 expression had a better prognosis than pathients with low levels.

Investigation of whether expression of bcl-2 influences the apoptotic response of doxorubicin-resistant breast cancer cells to a novel anthracycline

2082 Background: Doxorubicin is a mainstay of therapy for breast cancer patients. Recent studies have investigated the role of the anti-apoptotic protein bcl-2 in doxorubicin-resistance. Several reports describe that breast tumors with high levels of bcl-2 expression have decreased pathological and clinical responses to doxorubicin. WP744 is a doxorubicin analog which is benzylated at the C-4’ hydroxyl. We have previously shown that WP744 is superior to doxorubicin at inducing both growth inhibition and apoptosis of both doxorubicin-sensitive (MCF-Her-18) and doxorubicin-resistant (231-GFP, MCF-7) breast cancer cells. We now wished to determine the role of bcl-2 in the therapeutic response to WP744. Methods: MCF-Her-18, 231-GFP, and MCF-7 cells were incubated with PBS, doxorubicin (0.1 μg/ml), and WP744 (0.1 μg/ml). 231-GFP cells were transfected with vectors that overexpress bcl-2 (bcl-2 # 4, bcl-2 # 5) and a control vector (neu). At 72 h protein was isolated, subjected to gel electrophoresis, and transf...

Resveratrol-mediated sensitisation to TRAIL-induced apoptosis depends on death receptor and mitochondrial signalling

Natural food products such as resveratrol have gained considerable attention as cancer chemopreventive agents. In the present study, we investigated the potential of resveratrol to overcome the resistance of tumour cells against TRAIL. While resveratrol enhanced TRAIL-induced apoptosis through G1 cell cycle arrest and survivin depletion, resveratrol failed to sensitise cells with high expression levels of Bcl-2 or FADD-DN. Interestingly, overexpression of Bcl-2 or FADD-DN did not interfere with resveratrol-mediated cell cycle arrest or survivin depletion, but blocked release of cytochrome c and Smac from mitochondria into the cytosol, enhanced caspase activation and apoptosis upon combined treatment with resveratrol and TRAIL indicating that overexpression of Bcl-2 or FADD-DN decoupled the effect of resveratrol on the cell cycle and apoptosis. Similarly, cell cycle arrest at G1 using the cell cycle specific inhibitor mimosine or downregulation of survivin expression by antisense oligonucleotides failed to enhance TRAIL-induced apoptosis in Bcl-2- or FADD-DN-transfected cells. Likewise, inhibition of caspase activity using the broad range caspase inhibitor zVAD.fmk did not interfere with resveratrol-mediated cell cycle arrest and survivin depletion, while blocking apoptosis upon combined treatment with resveratrol and TRAIL. Thus, resveratrol is a potent sensitiser for TRAIL in certain tumours. However, it may be ineffective in others, e.g. in tumours with enhanced Bcl-2 expression or defective death receptor signalling.

P38 MAPK Mediates γ-Irradiation-induced Endothelial Cell Apoptosis, and Vascular Endothelial Growth Factor Protects Endothelial Cells through the Phosphoinositide 3-Kinase-Akt-Bcl-2 Pathway

Therapeutic radiation is widely used in cancer treatments. The success of radiation therapy depends not only on the radiosensitivity of tumor cells but also on the radiosensitivity of endothelial cells lining the tumor vasculature. Vascular endothelial growth factor (VEGF) plays a critical role in protecting endothelial cells against a number of antitumor agents including ionizing radiation. Strategies designed to overcome the survival advantage afforded to endothelial cells by VEGF might aid in enhancing the efficacy of radiation therapy. In this report we examined the signaling cascade(s) involved in VEGF-mediated protection of endothelial cells against gamma-irradiation. gamma-Irradiation-induced apoptosis of human dermal microvascular endothelial cells (HDMECs) was predominantly mediated through the p38 MAPK pathway as an inhibitor of p38 MAPK (PD169316), and dominant negative mutants of p38 MAPK could significantly enhance HDMEC survival against gamma-irradiation. Inhibition of the PI3K and MAPK pathways markedly up-regulated gamma-irradiation-mediated p38 MAPK activation resulting in enhanced HDMEC apoptosis. In contrast, VEGF-treated HDMECs were protected from gamma-irradiation-induced apoptosis predominantly through the PI3K/Akt pathway. Bcl-2 expression was markedly elevated in VEGF-treated HDMECs, and it was significantly inhibited by the PI3K inhibitor LY294002. HDMECs exposed to irradiation showed a significant decrease in Bcl-2 expression. In contrast, VEGF-stimulated HDMECs, when irradiated, maintained higher levels of Bcl-2 expression. Taken together our results suggest that gamma-irradiation induces endothelial cell apoptosis predominantly via the activation of p38 MAPK, and VEGF protects endothelial cells against gamma-irradiation predominantly via the PI3K-Akt-Bcl-2 signaling pathway.

Methioninase gene therapy with selenomethionine induces apoptosis in bcl-2-overproducing lung cancer cells.

We have previously shown that the toxic pro-oxidant methylselenol is released from selenomethionine (SeMET) by cancer cells transformed with the adenoviral methionine alpha,gamma-lyase (methioninase, MET) gene cloned from Pseudomonas putida. Methylselenol damaged the mitochondria via oxidative stress, and caused cytochrome c release into the cytosol thereby activating caspase enzymes and thereby apoptosis. However, gene therapy strategies are less effective if tumor cells overexpress the antiapoptotic mitochondrial protein bcl-2. In this study, we investigated whether rAdMET/SeMET was effective against bcl-2-overproducing A549 lung cancer cells. We established two clones of the human lung cancer A549 cell line that show moderate and high expression levels of bcl-2, respectively, compared to the parent cell line, which has very low bcl-2 expression. Staurosporine-induced apoptosis was inhibited in the bcl-2-overproducing clones as well as in the parental cell line. In contrast to staurosporine, apoptosis was induced in the bcl-2-overproducing clones as well as the parental cell line by AdMET/SeMET. Apoptosis in the rAdMET-SeMET-treated cells was determined by fragmentation of nuclei, and release of cytochrome c from mitochondria to the cytosol. A strong bystander effect of AdMET/SeMET was observed on A549 cells as well as the bcl-2-overproducing clones. rAdMET/SeMET prodrug gene therapy is therefore a promising novel strategy effective against bcl-2 overexpression, which has blocked other gene therapy strategies.

Bc1‐2 and MIB‐1 Labeling Indexes in Cats with Lymphoma

Immunohistochemistry was used to examine feline lymphoid tumors for bcl-2 and MIB-1 expression. Tumor tissues from 29 cats were selected to represent 2 groups--cats that did not respond to chemotherapy and cats that responded to therapy. Median bcl-2 immunoreactivity was 60%, and median MIB-1 reactivity was 47%. There was no significant difference in median survival time between cats with tumors with high levels of bcl-2 expression and those with low levels of expression. There was no significant difference in median survival time between cats with tumors with high levels of MIB-1 expression and those with low levels of expression. Mean bcl-2 immunoreactivity was significantly (P = .0004) higher in low-grade (73.2%) than in high-grade (16.9%) lymphomas, whereas the mean MIB-1 immunoreactivity was significantly (P = .0201) higher in high-grade (61.2%) lymphomas than in low-grade (35.0%) lymphomas. The mean bcl-2 immunoreactivity was significantly (P = .0042) greater in T-cell lymphomas (66.8%) than in B-cell lymphomas (22.8%), whereas the mean MIB-1 immunoreactivity was significantly (P = .0052) lower in T-cell lymphomas (36.4%) than in B-cell lymphomas (65.2%). Although expression of bcl-2 and MIB-1 did not appear to be linked to responses to chemotherapy in cats with lymphoma, the data suggest a possible role for these regulatory proteins in the biology of feline lymphomas.

Detection of BCL2 Rearrangements in Follicular Lymphoma

The identification and characterization of recurrent chromosomal translocations has improved tumor classification and yielded numerous insights into tumor pathogenesis, as such events typically lead to the dysregulation of proteins that control critical cellular processes, such as apoptosis, proliferation, differentiation, and immortalization. A prototypical example of such a chromosomal translocation is the (14;18)(q32;q32), which is highly associated with the B cell neoplasm follicular lymphoma. 1 Molecular analyses of DNA isolated from follicular lymphoma cells in the mid-1980’s showed that the t(14;18) creates a derivative chromosome 14 on which the BCL2 gene is juxtaposed to immunoglobulin heavy chain gene (IgH) sequences, 2-5 including enhancer sequences that override normal BCL2 gene control elements and drive inappropriately high levels of BCL2 expression in follicle center B cells. 6,7 Subsequently, several groups noted that enforced expression of BCL2 transgenes in murine hematopoietic cells promoted cell survival, 8,9 the first indication of the anti-apoptotic activity of BCL2. BCL2 transgenic mice also showed increased susceptibility to autoimmune disease 10 and B cell lymphoma, 11 observations that helped to foster the now generally accepted idea that dysregulation of apoptotic pathways is important in the pathogenesis of many forms of cancer and autoimmune disorders. These effects of BCL2 appear to be mediated through sequestration of “BH3 domain only” pro-apoptotic proteins such as BAD, BIM, and NOXA, 12 which resets the apoptotic “rheostat” toward increased resistance to programmed cell death. In addition to its impact on our thinking about cancer pathophysiology, the molecular characterization of the t(14;18) and its downstream consequences has influenced our approach to the diagnosis of hematological malignancy in several ways. The t(14;18) was an early example of an acquired genetic lesion that was strongly associated with a particular neoplasm, and thus of utility in tumor classification. Juxtaposed BCL2 and IgH DNA sequences provide a tumor-specific marker that can be exploited in the detection of minimal residual disease. 13,14 Furthermore, because BCL2 is down-regulated in normal germinal center B cells, 15 its expression (or lack thereof) can help to distinguish reactive and neoplastic follicles. 16 Finally, the presence of the t(14;18) has been suggested to be a marker of poor prognosis in diffuse large B cell lymphoma 17,18 (possibly because some such tumors arise through transformation of unrecognized underlying follicular lymphomas), although it must be said that more recent studies have not detected such a correlation. 19-21 These relationships have prompted the development of multiple methods that aim to detect the t(14;18) in tissue specimens. In this issue of The American Journal of Pathology, Albinger-Hegyi et al 22 report a new set of polymerase chain reaction (PCR)-based tests for detection of the BCL2/IgH fusion genes that offer some significant advantages in sensitivity over other more commonly used PCR-based methods. This commentary will attempt to place this contribution in context by discussing the advantages and limitations of various tests that aim to detect the t(14;18) or its downstream consequences.

Bcl-2 expression in F-MuLV-induced erythroleukemias: a role for the anti-apoptotic action of Bcl-2 during tumor progression

Erythroleukemias induced by various strains of Friend virus are multistage malignancies that result from the accumulation of genetic mutations, including the activation of proto-oncogenes and the inactivation of tumor suppressor genes. In this study, we demonstrate that Bcl-2 expression is activated in the majority of F-MuLV-induced erythroleukemia cell lines. In contrast, Bcl-2 was not expressed in any of the FV-P-induced erythroleukemia cell lines and protein levels were low or negligible in FV-A-induced erythroleukemia cell lines examined. In vivo, Bcl-2 expression levels gradually increased in F-MuLV-induced erythroleukemic cells prior to adaptation to culture. High expression of Bcl-2 in F-MuLV-induced erythroleukemic cells was shown to proceed the emergence of p53 mutation suggesting that Bcl-2 expression may delay p53 mutation in the leukemic cells. This is further supported by the demonstration that the majority of F-MuLV-induced erythroleukemia cell lines established from primary tumors induced in p53 mutant mice express low to negligible levels of Bcl-2. We have shown that the high levels of Bcl-2 expression in FV-P-induced erythroleukemic cells inhibited apoptosis induced by etoposide, low serum and p53 expression. Similarly, ectopic Bcl-2 expression within these cells also provided protection from apoptosis induced by etoposide and growth in low serum. These results suggest that the anti-apoptotic action of Bcl-2 may confer a selective in vivo and in vitro growth advantage to F-MuLV-induced erythroleukemic cells, which is not shared by FV-P/FV-A-induced erythroleukemic cells. The observed induction of Bcl-2 expression in vivo constitutes a novel but late oncogenic event associated with the progression of F-MuLV-induced erythroleukemias.

Prognostic significance of Ki-67 nuclear proliferative antigen, bcl-2 protein, and p53 expression in follicular and diffuse large B-cell lymphoma.

We analyzed 104 patients with non-Hodgkin's lymphoma, follicular or diffuse large-B-cell-type lymphoma, in order to evaluate the correlation between clinical characteristics and immunohistochemical parameters. Immunostaining was performed by means of monoclonal antibodies against Ki-67, bcl-2, and p53 expression. Forty-nine of the patients showed follicular lymphoma. A high expression of bcl-2 was found in 93%, high expression of p53 in 57%, and low expression of Ki-67 in 96%. Follicular lymphoma grade III showed a p53 expression (p = 0.07) slightly higher than follicular lymphoma grades I and II, not reaching statistical significance. Follicular lymphoma grades I and II tended to express lower Ki-67 and higher levels of bcl-2 expression than grade III (p = 0.06). Fifty-five cases showed diffuse large-B-cell lymphoma. Among them, bcl-2 was absent in 39%, whereas p53 and Ki-67 expression were high in 38%. In the diffuse large-B-cell lymphomas, a high bcl-2 expression correlated with stages III and IV (p = 0.03) and involvement of more than one extranodal area (p = 0.03). High Ki-67 expression was also associated to extranodal involvement of more than one area (p = 0.03). Overall survival of patients did not show statistically significant differences regarding Ki-67, bcl-2, and p53 tumoral expression. Prognostic factors for overall survival in the multivariate analysis were age (p = 0.02) and LDH (p = 0.003). Time to progression was worse among follicular lymphoma with high p53 expression than with mild/moderate p53 expression (p = 0.009).

Functional characterization of an IL-7–dependent CD4+CD8αα+ Th3-type malignant cell line derived from a patient with a cutaneous T-cell lymphoma

CDR3 of the functional rearranged T-cell receptor variable β region (TCR-Vβ) transcript was sequenced in order to demonstrate for the first time the identity between a long-term cultured T-cell line derived from a cutaneous T-cell lymphoma (CTCL) patient and the malignant T-cell clone present in the blood. The patient's peripheral blood lymphocyte-derived cultured T-cell line had a CD3+Vβ22+CD4+CD8+CD25−phenotype. It was named Pno and had been cultured for more than 1 year. Both fresh and long-term–cultured tumor cells proliferated highly in response to interleukin-7 (IL-7), and exogeneous IL-7 prevented Pno lymphocytes from apoptosis and maintained high levels of Bcl-2 expression. This unique malignant cloned lymphocyte line was further used to carry out functional studies. The results indicated that the CD3/TCR structures expressed by the Pno lymphocytes were functional because an immobilized anti-CD3 monoclonal antibody (mAb) or the combination of a soluble anti-CD3 mAb with submitogenic doses of phorbol 12 β-myristate 13 -acetate induced a proliferative response. Further, the CD2 and CD28 coreceptors were functional because they were able to induce a strong proliferative response upon their specific stimulation. Finally, the Pno T cell line had a Th3-type cytokine profile because it produced high amounts of the immunosuppressor cytokine tumor growth factor–β1 (TGF-β1). This high production of TGF-β1 may inhibit antitumor specific responses in CTCL.

Functional characterization of an IL-7–dependent CD4+CD8αα+ Th3-type malignant cell line derived from a patient with a cutaneous T-cell lymphoma

AbstractCDR3 of the functional rearranged T-cell receptor variable β region (TCR-Vβ) transcript was sequenced in order to demonstrate for the first time the identity between a long-term cultured T-cell line derived from a cutaneous T-cell lymphoma (CTCL) patient and the malignant T-cell clone present in the blood. The patient's peripheral blood lymphocyte-derived cultured T-cell line had a CD3+Vβ22+CD4+CD8αα+CD25−phenotype. It was named Pno and had been cultured for more than 1 year. Both fresh and long-term–cultured tumor cells proliferated highly in response to interleukin-7 (IL-7), and exogeneous IL-7 prevented Pno lymphocytes from apoptosis and maintained high levels of Bcl-2 expression. This unique malignant cloned lymphocyte line was further used to carry out functional studies. The results indicated that the CD3/TCR structures expressed by the Pno lymphocytes were functional because an immobilized anti-CD3 monoclonal antibody (mAb) or the combination of a soluble anti-CD3 mAb with submitogenic doses of phorbol 12 β-myristate 13 α-acetate induced a proliferative response. Further, the CD2 and CD28 coreceptors were functional because they were able to induce a strong proliferative response upon their specific stimulation. Finally, the Pno T cell line had a Th3-type cytokine profile because it produced high amounts of the immunosuppressor cytokine tumor growth factor–β1 (TGF-β1). This high production of TGF-β1 may inhibit antitumor specific responses in CTCL.