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PubMed1997

Apoptosis in the failing human heart.

BACKGROUND: Loss of myocytes is an important mechanism in the development of cardiac failure of either ischemic or nonischemic origin. However, whether programmed cell death (apoptosis) is implicated in the terminal stages of heart failure is not known. We therefore studied the magnitude of myocyte apoptosis in patients with intractable congestive heart failure. METHODS: Myocardial samples were obtained from the hearts of 36 patients who underwent cardiac transplantation and from the hearts of 3 patients who died soon after myocardial infarction. Samples from 11 normal hearts were used as controls. Apoptosis was evaluated histochemically, biochemically, and by a combination of histochemical analysis and confocal microscopy. The expression of two proto-oncogenes that influence apoptosis, BCL2 and BAX, was also determined. RESULTS: Heart failure was characterized morphologically by a 232-fold increase in myocyte apoptosis and biochemically by DNA laddering (an indicator of apoptosis). The histochemical demonstration of DNA-strand breaks in myocyte nuclei was coupled with the documentation of chromatin condensation and fragmentation by confocal microscopy. All these findings reflect apoptosis of myocytes. The percentage of myocytes labeled with BCL2 (which protects cells against apoptosis) was 1.8 times as high in the hearts of patients with cardiac failure as in the normal hearts, whereas labeling with BAX (which promotes apoptosis) remained constant. The near doubling of the expression of BCL2 in the cardiac tissue of patients with heart failure was confirmed by Western blotting. CONCLUSIONS: Programmed death of myocytes occurs in the decompensated human heart in spite of the enhanced expression of BCL2; this phenomenon may contribute to the progression of cardiac dysfunction.

Apoptosis↗

PubMed1999

BCL-2 blocks perforin-induced nuclear translocation of granzymes concomitant with protection against the nuclear events of apoptosis.

Cytolytic granule-mediated target cell killing is effected in part through the synergistic action of the membrane-acting protein perforin and serine proteases such as granzymes (Gr) A and B. In this study, we examine the subcellular distribution of granzymes in the presence of perforin and the induction of apoptosis in mouse FDC-P1 myeloid and YAC-1 lymphoma cells that express the proto-oncogene bcl2. Using confocal laser scanning microscopy to visualize and quantitate subcellular transport of fluoresceinated granzyme, we find that granzyme entry into the cytoplasm in the absence of perforin is not impaired in the bcl2-expressing lines. However, perforin-dependent enhancement of granzyme cellular uptake and, importantly, granzyme redistribution to the nucleus were strongly inhibited in the bcl2-expressing lines, concomitant with greatly increased resistance to granzyme/perforin-induced cell death. DNA fragmentation induced by granzyme/perforin was severely reduced in the bcl2-expressing lines, implying that prevention of granzyme nuclear translocation blocks the nuclear events of apoptosis. The kinetics of GrB nuclear uptake and induction of apoptosis were faster than for GrA, whereas YAC-1 cells showed greater resistance to granzyme nuclear uptake and apoptosis than FDC-P1 cells. In all cases, granzyme nuclear accumulation in the presence of perforin correlated precisely with ensuing apoptosis. All results supported the idea that GrA and GrB share a common, specific nuclear targeting pathway that contributes significantly to the nuclear changes of apoptosis.

Animals↗

PubMed2004

Molecular-cytogenetic characterization of non-Hodgkin's lymphoma with double and cryptic translocations of the immunoglobulin heavy chain gene.

The present study aimed to characterize the clinical and molecular-cytogenetic features of non-Hodgkin's lymphoma (NHL) with double translocation of the immunoglobulin heavy chain (IGH) gene. G-banding analysis, fluorescence in situ hybridization (FISH) with the IGH (Cgamma and VH) and oncogene (c-MYC, BCL1, BCL2, and BCL6) probes, and long-distance polymerase chain reaction (LD-PCR) were performed on 6 patients with B-cell lymphoma, one with angioimmunoblastic T-cell lymphoma, and one with acute lymphoblastic leukemia (ALL) with B-cell phenotype. G-banding analysis detected two different 14q32 translocations, t(14,18) and add (14)(q32) in a patient with ALL. Two distinct partners of double IGH translocation identified by FISH were as follows: c-MYC + BCL2 in 3 patients, c-MYC + BCL1 in 2, c-MYC + BCL6 in one, BCL2 + 9q22 in one, and 1q21 + 6q27 in one. Colocalization of BCL1 and c-MYC probes was demonstrated in a patient with mantle cell lymphoma. LD-PCR detected c-MYC/Cmu, c-MYC/Calpha and BCL6/Cmu, and c-MYC/Calpha fusion in each one patient. Seven of 8 patients showed high serum LDH. Central nervous system and leukemic involvement was observed in 5 and 6 patients, respectively. Median survival time of patients with c-MYC/IGH translocation was 9 months. The results defined a clinical subset of B-cell lymphoma/leukemia showing extremely poor prognosis. C-MYC/IGH translocation is possibly an evolutionary alteration following the primary IGH translocation with BCL1, BCL2, or BCL6. Furthermore, FISH identified one novel (9q22) and one cryptic chromosomal breakpoints (6q27) involved in IGH translocation.

Adult↗

PubMed2004

Pregnancy-induced alterations of B cell maturation and survival are differentially affected by Fas and Bcl-2, independently of BcR expression.

In the present work, we have analyzed the roles of two molecules involved in the regulation of cell survival, Bcl2 and Fas, in the pregnancy-induced down-regulation of B lymphopoiesis in mice. Our results show that the overexpression of the anti-apoptotic molecule Bcl2 in Bcl2-transgenic (Tg) B cells is able to protect 'D' fraction pre-B cells from pregnancy-induced deletion. In contrast, in Fas(lpr/lpr) mice bearing a mutated cell death receptor Fas, such B cell targets are not protected. Moreover, bone marrow B cell sub-populations at both ends of the differentiation pathway, i.e. pre-pro 'A' and mature 'E-F' fraction B cells, which are not the major targets of the pregnancy-induced down-regulation, are doubled during pregnancy in Fas(lpr/lpr) mice only. Altogether, these data strongly suggest that B cell down-regulation during pregnancy is due to apoptotic events blocked by Bcl2, but does not depend on a functional Fas receptor. The expression of a transgenic BcR in the 3-83mudelta BcR-Tg mouse model yields similar observations, which indicates that early BcR expression does not alter bone marrow B cell fates during pregnancy.

Animals↗

PubMed2001

Apoptosis can be a confusing factor in in vitro clastogenic assays.

Among the tests used to determine the mutagenic potential of chemicals, the chromosomal aberrations and micronucleus assays play an important role. These tests score either chromosomal structural aberrations at metaphase or micronuclei at interphase. One of the hallmarks of apoptosis is DNA fragmentation into 50-300 kpb leading to oligonucleosomal fragmentation that can interfere with the results of clastogenic assays. In this case, apoptosis may be a confusing factor in the evaluation of the mutagenic potential of molecules and lead to false positive results. For these reasons we have developed a cell line able to demonstrate the interference of apoptosis in two mutagenicity tests: the in vitro micronucleus test and metaphase analysis in vitro. We used a murine cytotoxic T cell line, CTLL-2 Bcl2, in which a stably transfected bcl2 gene is known to protect these cells from apoptosis induced by various stimuli. A comparison between results obtained in parental CTLL-2 cells and in CTLL-2 Bcl2 cells treated with non-genotoxic apoptosis inducers, such as dexamethasone or gliotoxin, leads us to conclude that apoptosis could give false positive results due to DNA fragmentation. Moreover, with etoposide, a clastogen that also induces apoptosis, we observed that the percentages of aberrant cells and numbers of micronuclei were significantly increased in CTLL-2 cells compared with CTLL-2 Bcl2 cells. This observation suggests that apoptosis leads to an overestimation of the genotoxic potential of chemicals. Finally, with nocodazole, an aneugen, we confirm that this model can also detect agents that have only genotoxic potential and thus allows a better estimation of the genotoxic threshold in studies with aneugens, thus avoiding overestimation of the mutagenic risk of such a compound.

Animals↗

PubMed2006

Effect of a vascular endothelial growth factor (VEGF) inhibitory treatment on the folliculogenesis and ovarian apoptosis in gonadotropin-treated prepubertal rats.

In the present study, we investigated whether vascular endothelial growth factor A (VEGFA) plays a critical intraovarian survival role in gonadotropin-dependent folliculogenesis. The effect of an intrabursal administration of a VEGFA antagonist on follicular development, apoptosis, and levels of pro- and antiapoptotic proteins of BCL2 family members (BAX, BCL2, and BCL2L1), as well as of TNFRSF6 (also known as FAS) and FAS ligand (FASLG), was examined. To inhibit VEGFA, a soluble FLT1/Fc Chimera (Trap) was administered to prepubertal eCG-treated rats. Injection of 0.5 mug of Trap per ovary did not change the number of preantral follicles (PFs) or early antral follicles (EAFs); however, it significantly decreased the number of periovulatory follicles 48 h after surgery and significantly increased the number of atretic follicles. No significant differences were found in any stage of the follicles either 12 or 24 h after injection. Cells undergoing DNA fragmentation were quantified by performing TUNEL on ovarian sections. Trap treatment caused a twofold increase in the number of apoptotic cells in EAFs. DNA isolated from antral follicles incubated for 24 h exhibited the typical apoptotic DNA pattern. Follicles obtained from Trap-treated ovaries showed a significant increase in the spontaneous onset of apoptotic DNA fragmentation. The injection of Trap significantly increased the levels of BAX and decreased the levels of BCL2 protein. The ratio of BCL2L1L:BCL2L1s was significantly diminished in follicles obtained from ovaries treated with Trap. No changes in the levels of TNFRSF6 or FASLG were observed after treatment. We concluded that the local inhibition of VEGFA activity appears to produce an increase in ovarian apoptosis through an imbalance among the BCL2 family members, thus leading a larger number of follicles to atresia.

Animals↗

PubMed2002

Expression of cell cycle-related gene products in Langerhans cell histiocytosis.

BACKGROUND The pathogenesis of Langerhans cell histiocytosis (LCH), a disease characterized by an abnormal accumulation of the dendritic Langerhans cells, is still unknown. Based on the monoclonality of the CD1a+ cell and reports of familial clustering, it is hypothesized that a genetic alteration at a cellular level may be causative. This genetic change may have an effect on the cellular mechanisms controlling proliferation and apoptosis. MATERIALS AND METHODS LCH-lesions were studied for the expression of Ki-67, present in the nucleus of proliferating cells. Furthermore, the expression of cell cycle-related gene products TGF-beta receptor I and II, MDM2, p53, p21, p16, Rb, and Bcl2 were studied. The TGF-betaR genes play a role in tumor suppression, whereas Bcl2 inhibits apoptosis. The remaining genes are part of either the p53-p21 and/or p16-Rb pathways, which induce cell cycle arrest or apoptosis in response to DNA damage. RESULTS In 30 biopsies the diagnosis of LCH could be confirmed on the basis of CD1a positivity (27 bone and 3 skin). All cases showed scattered nuclear-positive staining for the proliferation marker Ki-67. In more than 90% (n >/=27) of these cases, expression of TGFbeta receptor I and II, MDM2, p53, p21, p16, Rb, and Bcl2 was detected in lesional LCH cells. The overexpression was in general heterogeneous, ranging from limited focal staining of scattered cells within the lesion to strong diffuse staining. CONCLUSIONS These findings suggest that the cellular mechanisms that sense and respond to DNA-damage, namely the p53-p21 pathway and the p16-Rb pathway, are activated. The expression of Ki-67 indicates that the cells in LCH are proliferating. The observed overexpression of Bcl2 may play a role in the activation of p53 and p16 and/or the arrest of apoptosis.

Activin Receptors, Type I↗

PubMed2004

The effect of alpha-melanocyte-stimulating hormone on renal tubular cell apoptosis and tubulointerstitial fibrosis in cyclosporine A nephrotoxicity.

BACKGROUND: The pathogenesis of cyclosporine A (CsA)-induced nephrotoxicity has been known to be secondary to hemodynamic changes, but increasing evidence indicates that CsA has a direct toxicity to renal tubular cells, leading to their apoptosis and tubulointerstitial fibrosis. This study evaluated the mechanism for CsA-induced tubular cell apoptosis, tubulointerstitial fibrosis and its associated proteins, and the therapeutic effects of alpha-melanocyte-stimulating hormone (MSH) on them. METHODS: Male Sprague-Dawley rats fed with a low-sodium diet were divided into three treatment groups: group A (vehicle-injected group), group B (CsA 15 mg/kg-injected group), and group C(CsA+alpha-MSH-injected group). After 42 days, creatinine clearance; blood CsA level; apoptosis; inflammation and tubulointerstitial fibrosis in renal tissue; and the expression of Bax, Bcl2, Fas, FasL, and transforming growth factor (TGF)-beta protein were determined. RESULTS: CsA-induced tubular cell apoptosis; cellular infiltration; and increase of Fas, Bax, TGF-beta protein expression with significant tubulointerstitial fibrosis, and reduced Bcl2 protein expression. alpha-MSH treatment prevented the Bax and TGF-beta protein increase and induced Bcl2 protein increase, together with reduction of apoptosis, inflammation, and tubulointerstitial fibrosis. CONCLUSIONS: These findings suggest that chronic CsA nephrotoxicity is related to Bax and Bcl2-related apoptosis pathways, and that alpha-MSH can attenuate the CsA-induced tubulointerstitial fibrosis as well as tubular cell apoptosis.

Animals↗

PubMed2005

Transcription factor-mediated proliferation and apoptosis in benign and malignant thyroid lesions.

Transcription factors play an essential role in regulating both cell proliferation and programmed cell death. Proliferation and apoptosis-related transcription factor immunoexpression patterns were concomitantly investigated in tissue sections of normal thyroid, goiters, follicular adenomas and well-differentiated papillary and follicular carcinomas using antibodies against prothymosin alpha, E2F-1, p53, Bcl2, and Bax proteins. Proliferation and apoptotic indices were determined by Ki-67 immunoreactivity and the terminal deoxynucleotidyl transferase-mediated deoxy uridine triphosphate nick-end labeling technique, respectively. Prothymosin alpha and E2F-1 immunoexpression levels were found to be significantly elevated in well-differentiated carcinomas compared to adenomas, goiters and normal tissues (P < 0.05). Both proteins were directly correlated with the proliferation index (P < 0.05). E2F-1 was additionally correlated with the apoptotic index (P < 0.05). The majority of cases were negative for p53 staining. Positive Bcl2 immunostaining was detected in all thyroid histotypes. None of the normal tissues showed Bax immunoreactivity, while positive accumulation differed significantly between hyperplastic and neoplastic histotypes. Direct correlations were observed between prothymosin alpha and Bcl2 as well as between E2F-1 and Bax immunoexpression (P < 0.05). These data demonstrate that prothymosin alpha and E2F-1 are strongly involved in the proliferation processes of thyroid neoplasias. Furthermore, prothymosin alpha may promote cell survival through the Bcl2 anti-apoptotic pathway, while E2F-1-induced apoptosis via p53-independent pathways may be associated with transcriptional activation of bax pro-apoptotic gene.

Adenoma↗

PubMed2006

Involvement of caspase 1 and its activator Ipaf upstream of mitochondrial events in apoptosis.

PTP-S2/TC45 is a nuclear protein tyrosine phosphatase that activates p53 and induces caspase 1-dependent apoptosis. We analyzed the role of ICE protease-activating factor (Ipaf), an activator of caspase 1 in p53-dependent apoptosis. We also determined the sequence of events that lead to apoptosis upon caspase 1 activation by Ipaf. PTP-S2 expression induced Ipaf mRNA in MCF-7 cells which was dependent on p53. PTP-S2-induced apoptosis was inhibited by a dominant-negative mutant of Ipaf and also by an Ipaf-directed short-hairpin RNA. Doxorubicin-induced apoptosis was potentiated by the expression of caspase 1 (but not by a catalytic mutant of caspase 1) and required endogenous Ipaf. Doxorubicin treatment of MCF-7 cells resulted in activation of exogenous caspase 1, which was partly dependent on endogenous Ipaf. An activated form of Ipaf induced caspase 1-dependent apoptosis that was inhibited by Bcl2 and also by a dominant inhibitor of caspase 9 (caspase 9s). Caspase 1-dependent apoptosis induced by doxorubicin was also inhibited by Bcl2 and caspase 9s, but caspase 1 activation by activated Ipaf was not inhibited by Bcl2. Mitochondrial membrane permeabilization was induced by caspase 1 and activated Ipaf, which was inhibited by Bcl2, but not by caspase 9s. Expression of caspase 1 with activated Ipaf resulted in the activation of Bax at mitochondria. Our results suggest that Ipaf is involved in PTP-S2-induced apoptosis and that caspase 1, when activated by Ipaf, causes release of mitochondrial proteins (cytochrome c and Omi) through Bax activation, thereby functioning as an initiator caspase.

Animals↗

PubMed2004

Detection of methylated apoptosis-associated genes in urine sediments of bladder cancer patients.

PURPOSE: There is increasing evidence for a fundamental role for epigenetic silencing of apoptotic pathways in cancer. Changes in DNA methylation can be detected with a high degree of sensitivity, so we used the MethyLight assay to determine how methylation patterns of apoptosis-associated genes change during bladder carcinogenesis and whether DNA methylation could be detected in urine sediments. EXPERIMENTAL DESIGN: We analyzed the methylation status of the 5' regions of 12 apoptosis-associated genes (ARF, FADD, TNFRSF21, BAX, LITAF, DAPK, TMS-1, BCL2, RASSF1A, TERT, TNFRSF25, and EDNRB) in 18 bladder cancer cell lines, 127 bladder cancer samples, and 37 samples of adjacent normal bladder mucosa using the quantitative MethyLight assay. We also analyzed the methylation status in urine sediments of 20 cancer-free volunteers and 37 bladder cancer patients. RESULTS: The 5' regions of DAPK, BCL2, TERT, RASSFIA, and TNFRSF25 showed significant increases in methylation levels when compared with nonmalignant adjacent tissue (P < or = 0.01). Methylation levels of BCL2 were significantly associated with tumor staging and grading (P < or = 0.01), whereas methylation levels of RASSF1A and ARF were only associated with tumor stage (P < or = 0.04), and TERT methylation and EDNRB methylation were predictors of tumor grade (P < or = 0.02). To investigate clinical usefulness for noninvasive bladder cancer detection, we further analyzed the methylation status of the markers in urine samples of patients with bladder cancer. Methylation of DAPK, BCL2, and TERT in urine sediment DNA from bladder cancer patients was detected in the majority of samples (78%), whereas they were unmethylated in the urine sediment DNA from age-matched cancer-free individuals. CONCLUSIONS: Our results indicate that methylation of the 5' region of apoptosis-associated genes is a common finding in patients with bladder carcinoma. The ability to detect methylation not only in bladder tissue, but also in urine sediments, suggests that methylation markers are promising tools for noninvasive detection of bladder cancers. Our results also indicate that some methylation markers, such as those in regions of RASSF1A and TNFRSF25, might be of limited use for detection because they are also methylated in normal bladder tissues.

Adult↗

PubMed2006

A multimarker model to predict outcome in tamoxifen-treated breast cancer patients.

PURPOSE: This study was designed to produce a model to predict outcome in tamoxifen-treated breast cancer patients based on clinicopathologic features and multiple molecular markers. EXPERIMENTAL DESIGN: This was a retrospective study of 324 stage I to III female breast cancer patients treated with tamoxifen for whom standard clinicopathologic data and tumor tissue microarrays were available. Nine molecular markers were studied by semiquantitative immunohistochemistry and/or fluorescence in situ hybridization. Cox proportional hazards analysis was used to determine the contributions of each variable to disease-specific and overall survival, and machine learning was used to produce a model to predict patient outcome. RESULTS: On a univariate basis, the following features were significantly associated with worse survival: high pathologic tumor or nodal class, histologic grade, epidermal growth factor receptor, ERBB2, MYC, or TP53; absent estrogen receptor (ER) or progesterone receptor; and low BCL2. CCND1 and CDKN1B did not reach statistical significance. On a multivariate basis, nodal class, ER, and MYC were statistically significant as independent factors for survival. However, the benefit of ER-positive status was moderated by BCL2, ERBB2, and progesterone receptor. BCL2 and TP53 also interacted as an independent risk factor. A kernel partial least squares polynomial model was developed with an area under the receiver operating characteristic curve of 0.90. CONCLUSIONS: Our data show the predictive value of BCL2, ERBB2, MYC, and TP53 in addition to the standard hormone receptors and clinicopathologic features, and they show the importance of conditional interpretation of certain molecular markers. Our multimarker predictive model performed significantly better than standard guidelines.

Aged↗

PubMed2002

Follicular lymphoma with a novel t(14;18) breakpoint involving the immunoglobulin heavy chain switch mu region indicates an origin from germinal center B cells.

With the use of DNA-fiber fluorescent in situ hybridization, a BCL2 protein positive follicular lymphoma with a novel BCL2 breakpoint involving the immunoglobulin heavy chain (IGH) switch mu (S(mu)) region instead of the J(H) or D(H) gene segments was identified. Sequence analysis showed that the genomic breakpoint is localized between the S(mu) region of the IGH complex and the first intron of BCL2. Reverse-transcriptase polymerase chain reaction showed expression of a unique hybrid IGH-BCL2 transcript involving the transcription initiation site I(mu). Sequence analysis of the V(H) region of the functional nontranslocated IGH allele showed multiple shared somatic mutations but also a high intraclonal variation (53 differences in 15 clones), compatible with the lymphoma cells staying in or re-entering the germinal center. This is the first example of a t(14;18) translocation that results from an illegitimate IGH class-switch recombination during the germinal center B-cell stage.

Aged↗

PubMed2006

Prognostic impact of germinal center-associated proteins and chromosomal breakpoints in poor-risk diffuse large B-cell lymphoma.

PURPOSE: Outcome of diffuse large B-cell lymphoma (DLBCL) with a germinal center B-cell (GCB) expression profile is superior to that of non-GCB DLBCL. This conclusion is mainly derived from patients with mixed international prognostic index (IPI) risk profiles treated with CHOP-like therapy (cyclophosphamide, doxorubicin, vincristine, and prednisone). We wondered whether the prognostic impact of the expression profile would hold out in a homogeneous cohort of poor-risk DLBCL patients treated with high-dose sequential therapy (HDT) and autologous stem-cell transplantation (ASCT) as first-line therapy. PATIENTS AND METHODS: DLBCL from 66 newly diagnosed poor-risk patients, treated in two sequential prospective Dutch Hemato-Oncology Association (HOVON) trials, were studied retrospectively for expression of CD10, bcl6, MUM1/IRF4, bcl2, Ki67, and CD21+ follicular dendritic cells (FDC) by immunohistochemistry, and for the breakpoints of BCL2, BCL6, and MYC by fluorescent in situ hybridization (FISH). Lymphomas with any follicular component were excluded. RESULTS: A GCB immunophenotype profile was found in 58% and non-GCB immunophenotype profile in 42% of the tumors. Clinical characteristics of both groups were similar. Complete response (CR) rate was higher in patients with CD10+ tumors (58% v 30%; P = .03). A GCB immunophenotype profile, its constituting markers CD10 more than 30% and MUM1 less than 70%, and bcl2 less than 10% were each associated with a better overall survival (OS). FDC networks, equally present in GCB and non-GCB tumors, had superior CR (73% v 31%; P = .01), but disease-free survival rates were lower and there was no difference in OS rates. None of the breakpoints had a prognostic impact on outcome. CONCLUSION: Also in patients with poor-risk DLBCL treated with HDT and ASCT, the GCB immunophenotype and bcl2 expression retained a major impact on survival.

Adolescent↗

PubMed2005

Modulation of mitochondrial transition pore components by thyroid hormone.

Thyroid hormone (TH) modulates metabolic efficiency by controlling the coupling of mitochondrial oxidative phosphorylation. However, its uncoupling mode of action is still enigmatic. Treatment of Jurkat or GH3 cells with T3 is reported here to result in limited, Cyclosporin A-sensitive mitochondrial depolarization, conforming to low conductance gating of the mitochondrial transition pore (MTP). MTP protein components induced by T3 treatment were verified in T3-treated and hypothyroid rat liver as well as in Jurkat cells. T3 treatment resulted in increase in mitochondrial Bax and Bak together with decreased mitochondrial Bcl2. T3-induced mitochondrial depolarization was aborted by overexpression of Bcl2. In contrast to Bax-Bcl2 family proteins, some other MTP components were either not induced by T3 (e.g. voltage-dependent anion channel) or were induced, but were not involved in Cyclosporin A-sensitive MTP gating (e.g. Cyclophilin D and adenine nucleotide translocase-2) Hence, TH-induced mitochondrial uncoupling may be ascribed to low conductance MTP gating mediated by TH-induced increase in mitochondrial proapoptotic combined with a decrease in mitochondrial antiapoptotic proteins of the Bax-Bcl2 family.

Animals↗

PubMedDate not supplied

Coexpression of genes involved in apoptosis in central nervous system neoplasms.

PURPOSE: Apoptosis plays a crucial role in normal development and mediates tumor response to chemotherapy. This study investigated the pattern of apoptotic gene expression in brain tumor tissue specimens and cell lines. MATERIALS AND METHODS: BCL2, BCLXL, BCLXS, and BAX transcripts were amplified using reverse transcriptase polymerase chain reaction in 7 high-grade gliomas (HGGs), 7 ependymomas, and 6 cell lines (2 glioblastomas, 3 medulloblastomas, and 1 supratentorial-primitive neuroectodermal tumor [PNET]). Immunohistochemical staining for BCL2, BCLX, BAX, and p53 was performed in 7 pediatric low-grade gliomas (LGGs) and 7 pediatric HGGs. RESULTS: Six of seven gliomas, all ependymomas, and all glioblastoma and medulloblastoma cell lines expressed BCLXL and BAX. BCL2 expression was only detected in the supratentorial PNET line PFSK. BCLXS was absent in all tumors. By immunohistochemistry, no glial tumors stained positively for BCL2. Similar BAX and BCLX protein expression was observed in LGG and HGG. Three of five glioblastomas showed significant p53 expression. CONCLUSIONS: Coexpression of proapoptotic and antiapoptotic genes in human brain tumors supports the hypothesis that the relative expression of competing genes determines apoptotic threshold.

Apoptosis↗

PubMed1999

G2/M-phase arrest and death by apoptosis of HL60 cells irradiated with exponentially decreasing low-dose-rate gamma radiation.

Cells of the TP53-deficient human leukemia cell line HL60 continue to progress throughout the cell cycle and arrest in the G2/M phase during protracted exposure to exponentially decreasing low-dose-rate radiation. We have hypothesized that G2/M-phase arrest contributes to the extent of radiation-induced cell death by apoptosis as well as to overall cell killing. To test this hypothesis, we used caffeine and nocodazole to alter the duration of G2/M-phase arrest of HL60 cells exposed to exponentially decreasing low-dose-rate irradiation and measured the activity of G2/M-phase checkpoint proteins, redistribution of cells in the phases of the cell cycle, cell death by apoptosis, and overall survival after irradiation. The results from these experiments demonstrate that concomitant exposure of HL60 cells to caffeine (2 mM) during irradiation inhibited radiation-induced tyrosine 15 phosphorylation of the G2/M-phase transition checkpoint protein CDC2/p34 kinase and reduced G2/M-phase arrest by 40-46% compared to cells irradiated without caffeine. Radiation-induced apoptosis also decreased by 36-50% in cells treated with caffeine and radiation compared to cells treated with radiation alone. Radiation survival was significantly increased by exposure to caffeine. In contrast, prolongation of G2/M-phase arrest by pre-incubation with nocodazole enhanced radiation-induced apoptosis and overall radiation-induced cell killing. To further study the role of cell death by apoptosis in the response to exponentially decreasing low-dose-rate irradiation, HL60 cells were transfected with the BCL2 proto-oncogene. The extent of G2/M-phase arrest was similar for parental, neomycin-transfected control and BCL2-transfected cells during and after exponentially decreasing low-dose-rate irradiation. However, there were significant differences (P < 0.01) in the extent of radiation-induced apoptosis of parental and neomycin- and BCL2-transfected cells after irradiation, with significantly less radiation-induced apoptosis and higher overall survival in BCL2-transfected cells than similarly irradiated control cells. These data demonstrate that radiation-induced G2/M-phase arrest and subsequent induction of apoptosis play an important role in the response of HL60 cells to low-dose-rate irradiation and suggest that it may be possible to increase radiation-induced apoptosis by altering the extent of G2/M-phase arrest. These findings are clinically relevant and suggest a novel therapeutic strategy for increasing the efficacy of brachytherapy and radioimmunotherapy.

Apoptosis↗

PubMed1999

Comparative immunohistochemical and molecular analysis of uterine and extrauterine leiomyosarcomas.

Histologic criteria defining malignancy in smooth muscle tumors are currently site specific. This study was undertaken to determine whether, in leiomyosarcomas (LMS) occurring in different anatomic locations, there were differences in patterns of expression of molecules that have been demonstrated to be associated with biologically aggressive behavior in malignant neoplasms, and also to determine their diagnostic utility. Formalin-fixed paraffin-embedded tissue blocks were selected from 16 extrauterine leiomyosarcomas (EULMS), 14 cases of uterine leiomyosarcomas (ULMS) and from five cases each of uterine and extrauterine leiomyomas (LM). Utilizing immunohistochemical (ABC) techniques with antigen retrieval, we assessed serial sections of each tumor for immunoreactivity with Glut1, CD44s, bcl2, cyclin D1, and estrogen receptor. Molecular genotyping for detecting k-ras-2 point mutation, p53 gene loss, and mdm2 gene amplification was performed on microdissected tumor samples from the same histologic sections. All of the uterine and extrauterine LM were diffusely positive for CD44s, bcl2, and cyclin D1, and uniformly negative for Glut1. In contrast, 50% of the ULMS and 25% of EULMS were Glutl positive. Moreover, Glut1 positivity closely correlated with aggressive biologic behavior reflected by distant metastatic spread. Eighty-percent of LM and 70% of the ULMS were estrogen receptor positive, whereas only one retroperitoneal tumor had focal weak positivity. Over 80% of the extrauterine and 50% of the uterine sarcomas showed absence of CD44s immunoreactivity. Percentage of cyclin D1 immunoreactivity was independent of tumor grade and inversely proportional to the percent of bcl2 positivity. An LMS of the male breast contained k-ras-2 exon 1 point mutation (codon 12 aspartate substitution of glycine). P53 allelic imbalance was present in 29% of ULMS and 57% EULMS. Mdm2 amplification was present in three of six EULMS but not in ULMS. In addition to clinical staging, Glut1 positivity together with patterns of immunoreactivity of CD44 and bcl2 may be helpful in identifying aggressive smooth muscle tumors of the uterus and some EULMS. The presence of estrogen receptor staining may be helpful in identifying uterine versus nonuterine LMS. Although sample numbers are too small for definite conclusions, this study suggests that there are differences in glucose transport, expression of adhesion molecules, and estrogen receptors in ULMS and EULMS, which in part may be due to the estrogen dependency of the ULMS. P53 mutations and mdm2 amplifications appear to be more frequent in EULMS.

Adult↗