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PubMed2002

Liposome-mediated transfer of the bcl-2 gene results in neuroprotection after in vivo transient focal cerebral ischemia in an animal model.

Acute cerebral ischemia causes hypoxic neuronal cell death by necrosis and apoptosis. Expression of anti-apoptotic transgenes in ischemic brain may provide a useful therapeutic strategy for alleviation of postischemic damage. The present study investigates liposome-mediated transfer of the human bcl-2 protein in a rat model of focal transient ischemia due to middle cerebral artery (MCA) occlusion. Two different types of plasmid vectors were used for bcl-2 expression: one driven by the constitutive cytomegalovirus promoter (pCMV) and another based on the hypoxia-inducible human vascular endothelial growth factor promoter (pHRE). Cationic liposome/plasmid DNA complexes (lipoplexes) were injected directly into the cerebrospinal fluid (CSF) of rats immediately after MCA occlusion. The brains of treated and control animals were analyzed 48 h later. Infarct volumes and numbers of apoptotic cells were quantified. Occlusion of the MCA resulted in ipsilateral cerebral infarcts in all study animals. Transfer of the bcl-2 gene resulted in high level widespread protein expression in the case of the pCMV-bcl2 plasmid, while animals treated with the pHRE-bcl2 vector showed lower expression levels of bcl2 which were in addition limited to the ischemic area. Treatment with pCMV-bcl2, but not with pHRE-bcl2, was able to significantly reduce the infarct volume, which was 109 +/- 8 mm(3) for pCMV-bcl2, 152 +/- 29 mm(3) for pHRE-bcl2, and 155 +/- 18 mm(3) for control animals. Animals transfected with either vector showed a significant reduction in numbers of apoptotic cells in the infarct and penumbra area compared with controls. There were no short-term neurological side-effects of the CSF injection of lipoplexes or of bcl-2 expression. In conclusion, the hypoxia-inducible bcl-2 expression mediated by intrathecal lipoplexes may represent a novel, biologically safe and lesion-selective therapeutic approach for neuroprotection after acute cerebral ischemia. DOI: 10.1038/sj/gt/3301676

Acute Disease↗

PubMed2003

The t(14;18) is associated with germinal center-derived diffuse large B-cell lymphoma and is a strong predictor of outcome.

The t(14;18) is present in a significant proportion of diffuse large B-cell lymphomas (DLBCLs), however, the prognostic effect of the translocation and the relationship with transformed follicular lymphoma remains controversial. To clarify these uncertainties, interphase fluorescence in situ hybridization (FISH) was used to determine the incidence of the t(14;18) in nodal DLBCL, and this was correlated with BCL2 expression, germinal center (GC) immunophenotype, and patient outcome. FISH was performed on paraffin-extracted nuclei from 137 de novo nodal DLBCLs. Eighteen of 137 de novo DLBCLs were t(14;18) positive. The t(14;18) was most commonly associated with the subset of DLBCLs that expressed a GC phenotype, defined as CD10+, BCL6+ (GC-type DLBCL): positive in 14 of 47 (30%) cases, compared with 4 of 89 (5%) in the non-GC group (Pearson's chi(2) = 28.4; P < 0.0001). All cases with a translocation expressed BCL2 protein, however, 40 expressed BCL2 protein without a t(14;18). GC-type DLBCL patients with a t(14;18) had a significantly worse survival compared with GC-type DLBCL patients without the translocation (2-year survivals were 29 and 63%, respectively; P = 0.006). Of the cases without the translocation, BCL2 protein expression did not affect survival. In contrast, in the non-GC group of DLBCLs, BCL2 protein expression reduced the 2-year overall survival from 64% in the BCL2-negative group to 38%, with a median survival of 15.0 months (P = 0.02). In conclusion, the t(14;18) is common in DLBCLs, particularly in GC-type DLBCLs, where the presence of the translocation has a poor prognostic effect. BCL2 protein expression defines a group of non-GC DLBCL patients with a poor prognosis.

Chromosomes, Human, Pair 14↗

PubMed1998

Bax immunohistochemical expression in breast carcinoma: a study with long term follow-up.

Bax and Bcl2 are functionally antagonistic proteins which control apoptosis, whose expression in human tumours could be of prognostic value. We evaluated Bax and Bcl2 expression in 239 breast carcinomas (99 N0, 140 N1/2) with long term follow-up (median 79 months, range 11-140) in relation to clinico-pathologic parameters, clinical outcome, adjuvant therapy and expression of oestrogen receptor protein and p53. The prognostic value of Bax was investigated in the whole series of patients and in subgroups of homogeneously staged and treated patients (i.e., node-negative, N1/2 CMF-treated, N1/2 tamoxifen-treated). Bax immunostaining was cytoplasmic and heterogeneous. Cases were scored as Bax-positive if there were more than 20% reacting cells. High Bax expression was associated with positive nodal status (p = 0.03) and high Bcl2 expression (p = 0.01) and was more frequent in high-grade tumours. In the node-negative subgroup, Bax expression was associated with small tumour size. No association was seen with other parameters or with clinical outcome in any subgroup of patients. Since the apoptotic rate of a tumour is influenced by the ratio Bcl2/Bax, we investigated the combined effects of Bax and Bcl2 expression in relation to clinical outcome. However, no differences in survival were seen in the Bcl2-negative and Bcl2-positive groups when they were subdivided on the basis of the level of Bax expression and vice versa. In experimental systems, p53 is a direct transcriptional activator of the human bax gene. However, we could not observe any relation between Bax and p53 expression. We investigated whether the combined p53/Bax expression could have any prognostic value since it is predicted that tumours with normal p53 expression and concurrent high levels of Bax should be less aggressive and more susceptible to therapy. However, while p53 itself was of prognostic value, Bax expression was not related to prognosis in p53-negative or in p53-positive groups.

Breast Neoplasms↗

PubMed1996

Molecular heterogeneity of B-lineage diffuse large cell lymphoma.

B-lineage diffuse large cell lymphoma (B-DLCL) arising de novo is characterized by a marked degree of clinical heterogeneity. To determine whether or not the clinical heterogeneity of de novo B-DLCL is reflected by heterogeneity in the molecular features of these tumors, we investigated the pattern of distribution of several genetic lesions in 70 cases of de novo B-DLCL at diagnosis. The panel of genetic lesions tested comprised the molecular alterations most frequently detected in B-DLCL, including rearrangements of BCL2, BCL6, and MYC as well as deletions of 6q and mutations of TP53. One or more genetic lesions were detected in 39/70 cases of B-DLCL. Isolated structural alterations of BCL2, BCL6, 6q or TPS3 were detected in 8/70, 10/70, 11/70, and 3/70 cases, respectively. No isolated MYC lesions were detected. Six cases carried different combinations of two genetic lesions, including lesions of BCL2 + BCL6 (1 case), BCL2 + MYC (1 case), BCL2 + 6q (2 cases), or BCL6 + 6q (2 cases). One case had accumulated three genetic lesions, namely a rearrangement of BCL2 and BCL6 and a mutation of TPS3. Overall, these data show that multiple distinct patterns of genetic lesions may associate with de novo B-DLCL, indicating that the molecular pathogenesis of this group of lymphomas is characterized by a high degree of molecular heterogeneity.

Chromosome Deletion↗

PubMed2005

Variant t(14;18) in malignant lymphoma: a report of seven cases.

The BCL2 gene was identified through molecular analysis of the breakpoints involved in the t(14;18)(q32;q21) found in the majority of follicular lymphomas (FL). Variant translocations leading to juxtaposing of the BCL2 with either the IGK or IGL gene have been recognized in B-cell malignant lymphoma, although they are rare. We identified seven lymphoma cases that had variant translocations. Three cases had simple translocations involving two chromosomal regions: t(18;22)(q21;q11.2) in two cases and t(2;18)(p11.2;q21) in the third case. Complex translocations affecting more than two chromosomes were seen in the remaining four cases. Fluorescence in situ hybridization using the LSI IGH/BCL2 DNA probes revealed rearrangements of the BCL2 gene locus in all cases. In addition, expression of BCL2 protein was seen in all cases; only five of the seven cases expressed BCL6 protein. Morphologically, the lymphomas were categorized as B-cell follicular lymphoma in six cases and in the seventh case as diffuse large cell lymphoma (Richter syndrome) transformed from preexisting chronic lymphocytic leukemia (CLL). In case 2, the variant t(18;22) was seen as a secondary aberration evolving from a trisomy 12 clone. The findings revealed that BCL2 rearrangements in some malignant lymphomas occur through variant simple or complex chromosomal translocations, but always involving the IGH, IGK, or IGL chromosomal site. In addition, fluorescence in situ hybridization proved to be an important tool in evaluating these cases by showing IGH/BCL2 gene fusion or repositioning of the BCL2 gene.

Adult↗

PubMed1996

The p53-binding protein 53BP2 also interacts with Bc12 and impedes cell cycle progression at G2/M.

Using the yeast two-hybrid system, we have isolated a cDNA (designated BBP, for Bcl2-binding protein) for a protein (Bbp) that interacts with Bcl2. Bbp is identical to 53BP2, a partial clone of which was previously isolated in a two-hybrid screen for proteins that interact with p53. In this study, we show that specific interactions of Bbp/53BP2 with either Bcl2 or p53 require its ankyrin repeats and SH3 domain. These interactions can be reproduced in vitro with bacterially expressed fusion proteins, and competition experiments indicate that Bcl2 prevents p53 from binding to Bbp/53BP2. BBP/53BP2 mRNA is abundant in most cell lines examined, but the protein cannot be stably expressed in a variety of cell types by transfection. In transiently transfected cells, Bbp partially colocalizes with Bcl2 in the cytoplasm and results in an increased number of cells at G2/M, possibly accounting for the inability to obtain stable transfectants expressing the protein. These results demonstrate that a single protein can interact with either Bcl2 or p53 both in yeast cells and in vitro. The in vivo significance of these interactions and their potential consequences for cell cycle progression and cell death remain to be determined.

3T3 Cells↗

PubMed1999

Ascorbic acid inhibits apoptosis induced by X irradiation in HL60 myeloid leukemia cells.

Exposure of cells to ionizing radiation can cause apoptosis. Since antioxidants have been shown to protect against radiation-induced apoptosis, in this study we have evaluated the putative protective effect of ascorbate against radiation-induced apoptosis as well as the production of peroxides in the cells. HL60 cells transport the oxidized form of ascorbic acid, dehydroascorbic acid (DHA), and accumulate reduced ascorbate. Exposure of the cells to 5-40 Gy X radiation resulted in induction of apoptosis. Preincubation of the cells with DHA reduced the level of apoptosis after exposure to 5-20 Gy. Exposure of the cells to 5 or 20 Gy X radiation did not affect the intracellular concentration of peroxides, while phorbol myristate acetate (PMA), which is known to induce production of H(2)O(2) in cells (and served as a control), resulted in an increase in peroxides and a decrease in intracellular ascorbate. Irradiation of the cells with 1-3 Gy resulted in up-regulation of expression of BCL2 without affecting the level of apoptosis. At higher doses of radiation, enhanced BCL2 expression did not prevent radiation-induced apoptosis. Loading of the cells with ascorbate prior to their exposure to 1-3 Gy X radiation did not affect the enhanced BCL2 expression observed in the irradiated cells. At higher doses of radiation, ascorbate decreased apoptosis and restored the level of BCL2 in the cells. Exposure of the cells to 3-20 Gy X radiation enhanced the cell surface expression of TNFRSF6 (formerly known as Fas/APO-1) antigen and enhanced anti-TNFRSF6 antibody-induced apoptosis of the cells. Ascorbate loading did not affect expression of TNFRSF6 and did not overcome the anti-TNFRSF6 antibody-induced apoptosis. In conclusion, our data demonstrate that exposure of HL60 cells to radiation enhanced BCL2 and TNFRSF6 expression. Ascorbate did not affect BCL2 or TNFRSF6 expression. We therefore conclude that it protects HL60 cells against radiation-induced apoptosis, although the mechanisms of protection must still be elucidated.

Apoptosis↗

PubMedDate not supplied

A common pathway for chemotherapy-induced apoptosis in human squamous cell carcinoma lines distinct from that of receptor-mediated cell death.

Regulation of apoptosis by extracellular molecules binding to cell death receptors has received much attention in recent years. Fas, a member of the tumor necrosis factor receptor superfamily, is a transmembrane protein whose extracellular domain binds its cognate ligand (FasL), which can induce apoptosis in sensitive cells. Fas ligation leads to activation of cell death proteases, thereby initiating a proteolytic cascade which results in cellular fragmentation and death. Apoptosis is also regulated by inhibitory signals which promote cell survival. The bcl2 family of proteins is composed of both inhibitors and activators of programmed cell death. The bcl2 protein itself inhibits many apoptotic stimuli while other members of the bcl2 family such as bak and bid promote cell death. Many types of cancer chemotherapy induce cellular stress leading to induction of apoptosis. Stress-activated protein kinases such as p38 have been shown to inactivate bcl2 through phosphorylation and induce cleavage of bid. Deficiency of proapoptotic bcl2 family members has been associated with drug-resistant phenotypes. We report that exposure of human squamous cell carcinoma lines to different chemotherapy drugs activates a caspase cascade which is distinct from that of receptor-mediated apoptosis. The variable sensitivity of each cancer cell line to different forms of chemotherapy was not due to differences in caspase or bcl2 family protein expression. Rather, the stress-activated protein kinase p38 was overexpressed by resistant SCC lines which correlated with reductions in proapoptotic bid and bak protein expression. These two proteins exhibit distinct patterns of intracellular localization during chemotherapy-induced apoptosis.

Apoptosis↗

PubMed2005

Clinical, immunophenotypic, and genetic analysis of adult lymphomas with morphologic features of Burkitt lymphoma.

A prompt distinction of Burkitt lymphoma (BL) versus diffuse large B cell lymphomas (DLBCL) has important clinical implications; however, this distinction can be difficult. We analyzed 74 adult gray zone and 10 reference pediatric BL using immunohistochemistry (Ki-67, CD10, bcl2, bcl6) and fluorescence in situ hybridization (FISH) for MYC, BCL2, and BCL6 breakpoints. Two algorithms for classification were followed: algorithm A used a two-step review by four hemato-pathologists and algorithm B a set of only biologic markers (Ki-67 > or = 90%, CD10+, bcl6+, bcl2-, MYC breakpoint+, BCL2 and BCL6 breakpoint-). Both algorithms categorized all reference cases as BL. In the adult group, algorithm A resulted in 21 adult BL and 52 DLBCL and algorithm B in 23 BL and 51 "non-Burkitt" lymphomas (nBL); 9 cases (12%) contained two different translocations and were categorized as nBL in algorithm B. Fifteen cases (20%) fulfilled the BL criteria of both algorithms. Although not considered as BL according to both algorithms, many other lymphomas showed nonetheless a phenotypic and/or genetic shift to BL. BL according to algorithm B was more homogeneous with respect to clinical presentation (gender and localization) than BL defined by algorithm A. Our data suggest that only a few cases of these gray zone lymphomas represent true de novo BL. Immunohistochemistry for Ki-67, CD10, and bcl2 with analysis of MYC and preferably also BCL2 and BCL6 may be advised as a marker panel for this diagnostic dilemma.

Adolescent↗

PubMed2005

In vivo effects of highly active antiretroviral therapies containing the protease inhibitor nelfinavir on mitochondrially driven apoptosis.

BACKGROUND: In vitro studies have reported controversial effects of protease inhibitors (PIs) on mitochondrially driven apoptosis. Additionally, since PIs in the clinical setting are almost always given in combination with nucleoside analogues, which may have negative effects on mitochondrial DNA (mtDNA), the impact of PI-containing highly active antiretroviral therapy (HAART) on apoptosis and mtDNA content is unclear. PATIENTS AND METHODS: A cross-sectional study was performed including 20 HIV-negative (HIV-) patients, 16 HIV-positive, antiretroviral-naive (HIV+) patients and 17 HIV-positive patients receiving the PI nelfinavir (NFV) plus zidovudine and lamivudine (AZT+3TC) or didanosine and stavudine (ddl+d4T)--collectively known as HIV+PI--as first-line antiretroviral treatment for at least 12 months. Peripheral blood mononuclear cells (PBMCs) were isolated. BCL2 expression (anti-apoptotic) and the levels of the cleaved, active form of caspase-9 (pro-apoptotic) were determined by western blot. An index of mitochondrially driven apoptotic activation was estimated calculating the ratio caspase-9:BCL2. Mitochorldrial DNA content was measured by real-time PCR. RESULTS: BCL2 expression was lower in HIV+ than in HIV-patients (P < 0.01), whereas levels of caspase-9 were higher (P = 0.001). The caspase-9:BCL2 ratio was significantly increased in HIV+ compared with HIV-individuals (P < 0.001). Mitochondrial DNA content was also decreased in HIV+ compared with HIV-patients (P < 0.001). The HIV+PI group exhibited a trend to normalization for BCL2 expression and caspase-9 compared with the HIV+ group, whereas the caspase-9:BCL2 ratio significantly improved (decreased, P < 0.05 compared with HIV+ group). The mtDNA content in the HIV+PI group was similar to that of the HIV+ group, although the results of mtDNA content differed depending on whether NFV was combined with AZT+3TC (preserved) or with ddl+d4T (depleted). Conversely, no differences were found in apoptotic markers between the two subgroups of HIV+PI. CONCLUSIONS: NFV-based PI-containing HAART regimens may exert some beneficial effects counteracting the increased mitochondrially driven apoptosis present in HIV-infected people.

Adult↗

PubMedDate not supplied

Overexpression of BCL-x protein in primary breast cancer is associated with high tumor grade and nodal metastases.

PURPOSE: Dysregulation of genes that control apoptosis can contribute to tumor progression and increased drug resistance. The BCL2 gene and its family member BCL-x as well as the TP53 genes regulate apoptosis and have been shown to have a direct effect on the sensitivity of cancer cells to radiation and chemotherapeutic agents. METHODS: The expression of BCL-x, a BCL2-related protein that is a potent inhibitor of apoptosis, was investigated by immunohistochemical and immunoblot methods in 43 primary untreated breast carcinomas, in conjunction with BCL2 and TP53. RESULTS: BCL-x protein was overexpressed in 18 of 42 (43%) invasive breast cancers when compared with adjacent normal breast epithelium. Western blot analysis of eight primary breast cancers and five breast cancer cell lines indicated that BCL-xL was the predominant BCL-x protein expressed. Overexpression of BCL-x protein in these tumors was associated with higher tumor grade and increased number of positive nodes. In contrast, BCL2 protein was overexpressed in 19 of 42 tumors (45%) and was strongly correlated with estrogen receptor positivity, lower tumor grade, smaller tumor size, and lower stage. TP53 protein immunostaining was detected in 12 of 40 tumors (29%) and was inversely correlated with BCL2 expression and ER positivity. There was no correlation between the level of BCL-x protein expression and age, tumor size, ER status, and TP53 status. At a median follow-up time of 216 weeks, there was a trend toward decreased overall survival in patients with tumors overexpressing BCL-x. CONCLUSIONS: These findings suggest that expression of BCL-x protein is increased in a significant fraction of invasive breast cancers. In contrast to BCL2 expression, up-regulation of BCL-x protein may be a marker of tumor progression. Additional data including larger numbers of patients, more uniform treatments, and longer follow-up are needed to define the prognostic significance of overexpression of BCL-x during breast cancer progression.

Adult↗

PubMedDate not supplied

Degeneration of human oncogenes and mitochondrial genes occurs in cells that exhibit age-related pathology.

The development of a new class of assays to determine in vivo mutation frequencies has provided new perspectives on the timing, location, and distribution of somatic mutagenesis in mitochondrial genes and in oncogenes of the aging human body. This descriptive information has led to the inference of new models for age-related pathophysiology and oncogenesis. Mutations of mitochondrial genes rise rapidly with age to frequencies a thousand-fold higher than those of nuclear genes. Genotypic selection analysis has revealed that mitochondrial mutations accumulate predominantly in nonmitotic cells whose age-dependent loss is associated with pathology. Random mitochondrial mutation is most likely to inactivate Complex I, deficiency of which induces mitochondrial superoxide formation and cell death. Genotypic selection of oncogenic mutations at the BCL2 and p53 loci has revealed that the cell specificity of oncogenic mutations in persons without cancer correlates well with sites of tumor origin, indicating that cells bearing such mutations are the likely precursors of future tumors. Quantitative variation in human BCL2 mutation frequency is extensive, and BCL2 mutation frequency rises with age, concordant with increased risk for lymphoma. The clonality and persistence of BCL2 mutations suggests two specific testable mechanisms of lymphomagenesis. BCL2 mutation frequency rises in persons exposed to cigarette smoke, and more p53 mutations occur in skin exposed to sunlight than in unexposed skin. Thus, in addition to their likely relevance to future cancer risk, the dose-response relationship between exposure and oncogenic mutations indicates promise for their future use as in vivo biodosimeters of human exposure to carcinogens.

Aging↗

PubMed1995

Genotypic selection of mitochondrial and oncogenic mutations in human tissue suggests mechanisms of age-related pathophysiology.

The invention of the polymerase chain reaction (PCR) has facilitated the development of a new class of assays to quantify human somatic mutations in vivo, based on genotypic selection of mutants at the DNA level rather than phenotypic selection of mutants at the cell level. Use of these assays has provided new perspectives on the timing, location and distribution of somatic mutagenesis in mitochondrial genes and in oncogenes of the aging human body. This descriptive information has led to the inference and development of new models for age-related pathophysiology and oncogenesis. Mutations of mitochondrial genes rise rapidly with age to frequencies a thousand fold higher than those of nuclear genes. Genotypic selection analysis has revealed that mitochondrial mutations accumulate predominantly in non-mitotic cells whose age-dependent loss is associated with pathology. Random mitochondrial mutation is most likely to inactive Complex I, a deficiency of which induces mitochondrial superoxide formation and cell death. Genotypic selection of oncogenic mutations at the BCL2 and p53 loci has revealed that the cell specificity of oncogenic mutations in persons without cancer correlates well with sites of tumor origin, indicating that cells bearing such mutations are the likely precursors of future tumors. Quantitative variation in human BCL2 mutation frequency is extensive, and BCL2 mutation frequency rises with age, concordant with increased risk for lymphoma. The clonality and persistence of BCL2 mutations suggests two specific testable mechanisms of lymphomagenesis. BCL2 mutation frequency rises in persons exposed to cigarette smoke, and more p53 mutations occur in skin exposed to sunlight than in unexposed skin. Thus, in addition to their likely relevance to future cancer risk, the dose-response relationship between exposure and oncogenic mutations indicates promise for their future use as in vivo biodosimeters of human exposure to carcinogens.

Aging↗

PubMed2002

Alternative end-joining in follicular lymphomas' t(14;18) translocation.

T(14;18) chromosomal translocation is assumed to result from illegitimate rearrangement between the BCL2 proto-oncogene and the IGH locus during the D(H) to J(H) joining phase of V(D)J recombination in early B cells. Analysis of the breakpoint junctions suggests that translocation derives from the fusion between normal V(D)J recombination intermediates at the IGH locus and non-V(D)J-mediated broken-ends at the BCL2 locus. So far, BCL2 broken-ends have only been observed fused to coding-ends, raising questions concerning the molecular constraints of the illegitimate joining process. Using a combination of genome walking and long-range PCR assays, we describe in this report that in 4.5% (2/44) of the t(14;18), one of the BCL2 broken-ends is fused to a signal-end. The formation of these J(H)RSS/BCL2 junctions provides direct evidence that BCL2 broken-ends are capable of joining to both products of V(D)J recombination, suggesting their presence in the RAG-mediated post-cleavage complex. In addition, junctions generated by this alternative end-joining do not involve deletion of the chromosome 14 intervening sequences generally lost in the standard translocation, providing a unique opportunity to investigate the rearrangement status of this region in the translocated IGH allele. In both cases, a DJ(H) rearrangement could be detected 5' of the J(H)-RSS/BCL2 junction. These findings, together with the previously reported bias towards the most external D(H) and J(H) segments in standard breakpoints, strongly suggest that t(14;18) preferentially occurs during an attempted secondary D(H) to J(H) rearrangement. This unusual and restricted window of differentiation opens intriguing questions concerning the etiology of the translocation.

Base Sequence↗

PubMed2002

Immunohistochemical expression of CD10 and t(14;18) chromosomal translocation may be indicators of follicle centre cell origin in nodal diffuse large B-cell lymphoma.

AIMS: Although diffuse large B-cell lymphoma is categorized as a distinct entity in the REAL classification of lymphomas, it represents a heterogeneous group of neoplasms. A subgroup is probably of follicle centre cell origin and may evolve from a pre-existing follicular lymphoma. The t(14;18) chromosomal translocation can be demonstrated in the majority of follicular lymphomas and the aim of this study was to investigate the prevalence of t(14;18) translocation in a series of de novo nodal diffuse large B-cell lymphomas. We correlated this with the immunohistochemical expression of CD10, bcl2 and bcl6, markers which are usually expressed by the neoplastic cells in follicular lymphomas. We also correlated these parameters with the presence or absence of p53 protein expression by the neoplastic cells. METHODS AND RESULTS: Nodal diffuse large B-cell lymphomas (n=34) were stained immunohistochemically with monoclonal antibodies to CD10, bcl2, bcl6 and p53 (D07). Polymerase chain reaction (PCR) for the t(14;18) translocation was also performed. Fourteen, 24 and 29 (41%, 71%, 85%) cases exhibited positivity for CD10, bcl2 and bcl6, respectively. In 12 cases there was positivity with D07 (35%). By PCR, the t(14;18) translocation was identified in five cases (15%), four of which were positive for CD10 and bcl2 and all of which were positive for bcl6. One of five cases positive for the chromosomal translocation exhibited positivity with D07. CONCLUSIONS: In this study the t(14;18) translocation was identified in 15% of diffuse large B-cell lymphomas, all but one of which exhibited positivity for CD10, bcl2 and bcl6. These may represent cases of follicle centre cell origin which may or may not have evolved from a pre-existing follicular lymphoma. It is possible that positivity for CD10 especially may identify cases which are of follicle centre cell origin and that the absence of t(14;18) translocation in some of these cases may reflect the fact that the translocation cannot normally be demonstrated in all follicular lymphomas. Whether the presence or absence of the translocation and the immunophenotype are prognostically important should be investigated further.

Biomarkers, Tumor↗

PubMed2000

Expression of apoptosis regulatory proteins in tubular epithelium stressed in culture or following acute renal failure.

BACKGROUND: While tubular cell death is a characteristic of acute renal failure (ARF), the molecular mechanisms that modulate this cell death are unclear. Cell fate in acute renal failure hinges on a balance of survival and mortality factors in a changing environment. We further explored this issue by studying selected cell death-related proteins in experimental renal failure. METHOD: The expression of genes that promote (c-myc, Bax, BclxS) or protect (Bcl2, BclxL) from cell death was studied by Northern blot, Western blot, and immunohistochemistry in murine kidneys following ARF induced by folic acid or in renal tubular epithelial cells (MCT) stressed in culture. RESULTS: Renal mRNA levels encoding for c-myc and BclxL were elevated in ARF while the Bcl2/Bax ratio was decreased (Bcl2 decreased and Bax increased; P < 0.05). Protein levels of BclxL increased and Bcl2 protein decreased. Expression of tumor necrosis factor (TNF-alpha), a mediator of ARF, was also increased. Immunohistochemistry further demonstrated that BclxL was increased in some tubuli and absent in others, while Bcl2 expression decreased diffusely. Bax staining was also patchy among tubuli and individual cells in the tubular wall and lumen. As a relative deficit of survival factors is present in ARF, MCT epithelium were deprived of serum survival factors. This resulted in apoptosis, decreased Bcl2/Bax and BclxL/Bax ratios (P < 0.05) and sensitization to TNF-alpha-induced apoptosis (P < 0.05). The latter was prevented by enforced overexpression of BclxL (P < 0.01). TNF-alpha increased the mRNA levels encoding for c-myc and decreased BclxL expression. Neither MCT cells nor the kidney expressed BclxS. CONCLUSIONS: A relative deficit of survival factors likely contributes to changes in levels of BclxL and Bax in ARF. These deficits predispose to cell death induced by persistent lethal factors such as TNF-alpha that is increased in ARF and a potential source of increased c-myc, a downstream facilitator of cell death. These findings implicate members of the Bcl2 family of proteins as regulators of tubular cell death in ARF and single them out as potential therapeutic targets.

Acute Kidney Injury↗

PubMed2003

Hypoxia-induced apoptosis in cultured glomerular endothelial cells: involvement of mitochondrial pathways.

BACKGROUND: Glomerular endothelial cells (GENs) play a key role in the preservation and reconstruction of the glomerular capillary network following injury, thus maintaining the tissue oxygenation. Accumulating evidence has shown that failure to maintain the microcirculation leads to irreversible glomerular injury and glomerular sclerosis. In this regard, the behavior of endothelial cells in a hypoxic milieu is of interest. METHODS: We exposed cultured GENs to hypoxia and observed apoptosis by annexin V assay. We examined mitochondrial signaling, focusing on Bcl2 and Bax by real-time polymerase chain reaction (PCR), immunocytochemistry, and immunoprecipitation. Furthermore, we examined the response to hypoxia in an overexpression model of Bcl2. RESULTS: Hypoxic treatment induced apoptosis in 12.8%+/- 1.1% of GENs at 24 hours, and in 19.8%+/- 0.9% at 24 hours followed by 8 hours of reoxygenation. The expression of Bcl2 mRNA decreased to 0.45- +/- 0.15-fold at 24 hours, whereas that of Bax increased to 7.3- +/- 1.2-fold 1 hour after reoxygenation, accompanied by translocation from the cytosol to mitochondria. These changes were associated with a decrease in mitochondrial membrane potentials and an increase in caspase-9 activity. Both overexpression of Bcl2 and inhibition of Bax protected GENs from hypoxic injury. CONCLUSION: We conclude that changes of quantity and localization of Bcl2 and Bax contribute to hypoxia-mediated apoptosis of GENs in vitro. Further investigation into glomerular endothelial cell injury and intracellular signaling in a hypoxic milieu is required to better understand and ultimately prevent progression of chronic kidney disease.

Animals↗

PubMed1998

The relationship between BcI2, Bax and p53: consequences for cell cycle progression and cell death.

Each cell is under constant surveillance to maintain the integrity of its genome. Genomic lesions in a cell must be repaired before the onset of DNA replication and cell division. In the scenario that the genomic lesion is not repairable, the damaged cells are disposed in an orderly manner known as programmed cell death or apoptosis. Apoptosis and cell cycle progression are two intimately linked phenomena. Uncontrollable cell proliferation perturbs the cellular homeostasis and this can lead to malignancies, as well as organ dysfunction and developmental abnormalities. The biological pathway controlling cell fate is sequentially organized at the molecular level. Recent studies have made important contributions in advancing our knowledge of the mechanisms of cell cycle control and apoptosis regulation. A oncogene-derived protein, Bcl2, confers negative control in the pathway of cellular suicide machinery. A Bcl2-homologous protein, Bax, promotes cell death by competing with Bcl2. While Bax-Bax homodimers act as apoptosis inducers, Bcl2-Bax heterodimer formation evokes a survival signal for the cells. Both Bcl2 and Bax are transcriptional targets for the tumour suppressor protein, p53, which induces cell cycle arrest or apoptosis in response to DNA damage. In all, the coordinate performance of these molecules is crucial for controlling life and death of a cell.

Animals↗