Biomedical subjects

S Krajewski

Publications and source records attributed to S Krajewski.

At least 91 records · Page 5Linked to original sources

PubMed1997

Immunohistochemical analysis of in vivo patterns of TRAF-3 expression, a member of the TNF receptor-associated factor family.

An immunohistochemical approach was used to explore the in vivo expression of TNF receptor-associated factor 3 (TRAF-3), a putative signaling protein that binds to the cytosolic domains of CD30, CD40, and lymphotoxin-beta receptors. TRAF-3 immunostaining was detected in many types of cells throughout the human body. TRAF-3 immunostaining was only rarely present in thymocytes but was found in the thymic epithelioreticular cells. Lymphocytes in the bone marrow were also typically TRAF-3 immunonegative, whereas myeloid progenitor cells and megakaryocytes were often TRAF-3 positive. Peripheral blood lymphocytes were mostly TRAF-3 immunonegative, while granulocytes were TRAF-3 immunopositive. Monocytes were strongly immunostained for TRAF-3, but macrophages in nodes typically contained little or no TRAF-3 immunoreactivity. Some lymphocytes within the germinal centers of secondary lymphoid follicles in normal and reactive nodes were TRAF-3 immunopositive, as were occasional interfollicular lymphocytes in the T cell regions of these organs, but most lymphocytes appeared to be TRAF-3 immunonegative or stained only weakly. Plasma cells, however, were strongly TRAF-3 positive. Stimulation of PBLs with anti-CD3 Ab induced marked increases in the steady state levels of TRAF-3 protein in vitro as determined by immunoblotting, while levels of TRAF-2 were unchanged, implying a dynamic regulation of TRAF-3 expression. The findings establish for the first time the cell type- and differentiation-specific patterns of expression of a member of the TRAF family of proteins.

Amino Acid Sequence↗

PubMed1997

Interferon-gamma protects against herpes simplex virus type 1-mediated neuronal death.

Host inflammatory mediators, such as interferons, play a protective role in infection, but the mechanism is undefined and may differ between tissue compartments. To determine whether interferon-gamma (IFN-gamma) elicitation prevents destructive encephalitis in herpes simplex virus type 1 (HSV-1) infection of the central nervous system, IFN-gamma-knockout (GKO) mice were challenged intravitreally with HSV-1 strain F, inciting infection of the eyes and the brain. Indeed, the GKO mice showed encephalitis with ataxia, whereas nontransgenic controls remained asymptomatic. Morphology and digoxigenin labeling of DNA fragments revealed increased apoptosis in the brains of GKO mice compared with controls, although viral replication was not influenced at early stages of infection. Greater numbers of apoptotic cells in the brains of GKO mice correlated with neurological symptoms, as well as lower expression of the protective protooncogene bcl-2. Thus, IFN-gamma inhibits apoptosis, affording neuronal protection from destructive encephalitis during viral infection of the central nervous system.

Animals↗

PubMed1997

Apoptosis in gastric epithelial cells is induced by Helicobacter pylori and accompanied by increased expression of BAK.

Carriage of the bacterium H. pylori in the human stomach is associated with evidence of increased epithelial cell apoptosis. This may be of significance in the etiology of gastritis, peptic ulcers, and neoplasia. The ability of H. pylori to directly induce epithelial apoptosis was examined in vitro by fluorescence and electron microscopy, flow cytometry, and DNA fragmentation ELISA. The induction of apoptosis by H. pylori was time and concentration-dependent and inhibited by preventing direct bacterial-epithelial cell contact. Apoptosis was accompanied by increased expression of Bak, with little change in expression of other Bcl-2 family proteins. The expression of Bak was also increased in gastric biopsies from patients colonized by H. pylori. Thus, H. pylori induces gastric epithelial cell apoptosis, by a Bak-dependent pathway.

Apoptosis↗

PubMed1997

Prognostic significance of Bax protein expression in diffuse aggressive non-Hodgkin's lymphoma.

Bax is a proapoptotic member of the Bcl-2 protein family. The incidence and prognostic significance of Bax protein expression in diffuse non-Hodgkin's lymphomas with a large cell component (DLCL) was determined by an immunohistochemical method by using paraffin-embedded tumors from a cohort of patients treated uniformly with combination chemotherapy (n = 139). All patients were between 16 and 70 years of age and had advanced stage disease of diffuse large cell type (diffuse mixed, diffuse large cell, immunoblastic, or anaplastic large cell). Paraffin sections from diagnostic biopsies were successfully immunostained for Bax in 113 cases. Of these, 7 (6%) tumors were scored as Bax immunonegative (< 1% Bax-stained tumor cells), 42 (37%) as low (1% to 10%), 9 (8%) as low-intermediate (11% to 30%), 25 (22%) as high-intermediate (31% to 70%), and 30 specimens (27%) as high for Bax expression (> 70%). Of the 7 Bax-immunonegative lymphomas, all also scored low (< or = 10% immunostained tumor cells) for Bcl-2 expression, whereas 78 of the 106 (74%) Bax-immunopositive tumors had low Bcl-2 expression. By itself, Bax expression was not of prognostic significance in univariate analysis, although there was a clear trend for patients with Bax-immunonegative lymphomas (n = 7) to relapse sooner and to die faster than patients whose tumors contained Bax-immunopositive malignant cells (n = 106; 8-year overall survival 29% versus 55%; P = .06). When combined with Bcl-2 immunostaining data, Bax provided additional prognostic information. Among patients with Bcl-2 low-expressing DLCLs, for example, Bax immunonegativity was associated with lower 8-year relapse-free survival (RFS; 29% v 61%; P < .01) and lower 8-year overall survival (OS; 29% v 63%; P < .05), suggesting that absence of Bax protein connotes a more aggressive phenotype when Bcl-2 protein is also not expressed at high levels. In contrast, low Bax expression was associated with improved 8-year disease-free survival (52% v 16%; P < .02), RFS (47% v 11%; P < .02), and OS (64% v 11%; P < .01) in patients whose tumors expressed Bcl-2 at high levels, suggesting that the combination of high levels of Bax and Bcl-2 expression is more deleterious than high levels of Bcl-2 expression alone. Bax expression failed to provide additional prognostic information beyond Bcl-2 expression in multivariate analysis that included the clinical International Prognostic Index factors (age, stage, lactate dehydrogenase, performance status, and number of extranodal sites) and immunophenotype. Taken together, the results suggest that Bax expression is not a major prognostic marker in DLCL. However, the interactions of the Bcl-2 and Bax expression data with respect to clinical outcome may shed new insights into the biological significance of Bcl-2/Bax protein heterodimerization.

Adolescent↗

PubMed1997

Immunohistochemical analysis of interleukin-1beta-converting enzyme/Ced-3 family protease, CPP32/Yama/Caspase-3, in Hodgkin's disease.

The Caenorhabditis elegans cell death gene, Ced-3, encodes a protein homologous to mammalian interleukin-1beta-converting enzyme (ICE), a cysteine protease implicated in programmed cell death (PCD). CPP32, also known as Yama, apopain, and Caspase-3, is a member of this family, has substrate specificities similar to Ced-3, and has been shown to have an active role in PCD. Evidence suggests that these proteases act downstream of inhibitors of PCD such as Bcl-2 and Bcl-x(L), which are frequently expressed in Reed-Sternberg (RS) cells of Hodgkin's disease (HD). To date there have been no studies examining the role of the ICE/Ced-3 family of proteins, in particular CPP32, in HD. We examined 24 cases of HD with a classical immunophenotype and 6 cases of nodular lymphocyte predominant HD (NLPHD) for the expression of CPP32 in the RS cells and lymphohistiocytic (L&H) cells as detected by immunohistochemistry. Twenty two of 24 cases (92%) of HD expressed the protein in the RS cells, whereas the L&H cells in all 6 cases of NLPHD lacked expression of CPP32. These results provide further evidence that NLPHD is a phenotypically different disease distinct from classical forms of HD. The differential expression of the cell death protein CPP32 may be an important factor contributing to the apparently different clinical behaviour of NLPHD in contrast to classical HD. The lack of expression of CPP32 in NLPHD shares similarities with low-grade B-cell non-Hodgkin's lymphomas and may explain their common clinical course. Further studies are required to elucidate the significance of CPP32 in HD.

Caspase 3↗

PubMed1997

bcl-2 and bak may play a pivotal role in sodium butyrate-induced apoptosis in colonic epithelial cells; however overexpression of bcl-2 does not protect against bak-mediated apoptosis.

Butyrate, a short chain fatty acid produced in the colon as a result of fermentation of dietary fibre by symbiotic bacteria, induces apoptosis in colonic tumour cell lines. Three human colonic adenoma cell lines (AA/C1, RG/C2 and BH/C1) and one carcinoma cell line (S/KS/FI) were used to determine the effects of butyrate on the expression of bcl-2, bax and bak to examine the possible role of these proteins in the induction of apoptosis. RG/C2 and BH/C1 cells express p-26-bcl-2 and butyrate treatment decreased p26-bcl-2 levels in association with apoptosis, whereas bax and bak levels remained constant. AA/C1 and S/KS/FI cells have no detectable p26-bcl-2. In S/KS/FI cells, bax or bak levels did not change in response to butyrate. However, in AA/C1 cells, butyrate-induced apoptosis was associated with increased bak levels. Therefore, in AA/C1 cells butyrate-induced apoptosis appears to be mediated through bak. Furthermore, butyrate also induced apoptosis and increased bak levels in AA/C1 cells transfected with a bcl-2 expression vector which expressed high levels of p26-bcl-2. For S/KS/FI cells, two bcl-2 transfectants gave different results. bcl-2 protected against apoptosis in one transfectant in which bak levels were not elevated in response to butyrate, whereas it did not protect in the other transfectant in which bak levels were increased after butyrate treatment. The results suggest that expression of constitutively high levels of p26-bcl-2 only conferred protection against apoptosis when bak levels were not elevated in response to butyrate and that expression of constitutively high levels of p26-bcl-2 does not counter the effects of bak. Different mechanisms appear to be involved in cell death signalling in different tumours since butyrate may induce apoptosis via elevated levels of bak or reduced levels of p26-bcl-2.

Adenoma↗

PubMed1997

Prognostic significance of Bcl-2 protein expression and Bcl-2 gene rearrangement in diffuse aggressive non-Hodgkin's lymphoma.

The prognostic significance of Bcl-2 protein expression and bcl-2 gene rearrangement in diffuse large cell lymphomas (DLCL) is controversial. Bcl-2 protein expression prevents apoptosis and may have an important role in clinical drug resistance. The presence of a bcl-2 gene rearrangement in de novo DLCL suggests a possible follicle center cell origin and perhaps a distinct clinical behavior more akin to low-grade non-Hodgkin's lymphoma (NHL). The purpose of this study was to determine the impact of Bcl-2 protein expression and bcl-2 gene rearrangement (mbr and mcr) on survival of a cohort of patients with DLCL who were uniformly evaluated and treated with effective chemotherapy. Patients included the original MACOP-B cohort (n = 121) and the initial 18 patients treated with the VACOP-B regimen (total = 139). All patients had advanced-stage disease, were 16 to 70 years old, and corresponded to Working Formulation categories F, G, or H. No patients had prior treatment, discordant lymphoma, or human immunodeficiency virus seropositivity. Paraffin sections from diagnostic biopsies were analyzed for bcl-2 gene rearrangement including mbr and mcr breakpoints by polymerase chain reaction and Bcl-2 protein expression by immunohistochemistry. With a median follow-up of 81 months, overall (OS), disease-free (DFS), and relapse-free survival (RFS) were measured to determine the prognostic significance of these parameters. Analyzable DNA was present in 118 of 139 (85%) cases, with 14 demonstrating a bcl-2 rearrangement (11 mbr, 3 mcr). All 14 of these bcl-2 gene rearrangement-positive cases were found in the 102 patients with a B-cell immunophenotype, but the presence of this rearrangement had no significant influence on survival. Bcl-2 protein expression was interpretable in 116 of 139 (83%) cases, with immunopositivity detected in 54 of 116 (47%). Using a cut-off of greater than 10% Bcl-2 immunopositive tumor cells for analysis, positive Bcl-2 protein expression was seen in 28 of 116 (24%) patients and the presence of this expression correlated with decreased 8-year OS (34% v 60%, P < .01), DFS (32% v 66%, P < .001), and RFS (25% v 59%, P < .001). Bcl-2 protein expression remained significant in multivariate analysis that included the clinical international prognostic index factors and immunophenotype (P < .02). In conclusion, although bcl-2 gene rearrangement status could not be shown to have an impact on outcome, Bcl-2 protein expression is a strong significant predictor of OS, DFS, and RFS in DLCLs.

Adolescent↗

PubMed1997

Interleukin-13 in combination with CD40 ligand potently inhibits apoptosis in human B lymphocytes: upregulation of Bcl-xL and Mcl-1.

Interleukin-13 (IL-13) is a novel T-cell-derived cytokine with IL-4-like effects on many cell types. In human B lymphocytes, IL-13 induces activation, stimulates proliferation in combination with anti-IgM or anti-CD40 antibodies, and directs Ig isotype switching towards IgE and IgG4 isotypes. We show here that IL-13 also regulates human B-cell apoptosis. IL-13 reduced spontaneous apoptosis of peripheral blood B cells in vitro, as shown by measurement of DNA fragmentation using the TUNEL and Nicoletti assays. The inhibition of cell death by IL-13 alone was significant but modest, but was potently enhanced in combination with CD40 ligand (CD40L), a survival stimulus for B cells by itself. Interestingly, IL-13 increased the expression of CD40 on peripheral blood B cells, providing a possible mechanism for the observed synergy. IL-13 alone was a less potent inhibitor of apoptosis than IL-4. Moreover, there was no additive effect of combining IL-4 and IL-13 at supraoptimal concentrations, which is consistent with the notion that the IL-4 and IL-13 binding sites share a common signaling subunit. The combination of IL-13 with CD40L augmented the expression of the Bcl-2 homologues Bcl-xL and Mcl-1, suggesting this as a possible intracellular mechanism of induced survival. By contrast, levels of Bcl-2, and two other Bcl-2 family members, Bax and Bak, remained unaltered. Given the importance of the CD40-CD40L interaction in B-cell responses, these results suggest a significant role of IL-13 in the regulation of B-cell apoptosis.

Apoptosis↗

PubMed1997

Bax accelerates staurosporine-induced but suppresses nigericin-induced neuronal cell death.

Bax, a member of the Bcl-2 multigene family, is known to promote apoptosis. To investigate the role of Bax in an experimentally induced cell death of the murine dopaminergic neuronal cell line (MN9D), we established MN9D cells stably over-expressing murine Bax (MN9D/ Bax) or vector alone (MN9D/Neo). In MN9D/Neo cells treated with either 1 microM staurosporine or 0.1 microM nigericin, a ladder pattern of DNA fragmentation was induced. As expected, over-expression of Bax in MN9D cells accelerated staurosporine-induced cell death as measured by the MTT reduction assay (62.3% survival in MN9D/Neo vs 27.0% survival in MN9D/Bax). Surprizingly, both nigericin-induced cell death and its accompanying DNA fragmentation were largely attenuated in MN9D/Bax cells (22.0% survival in MN9D/Neo vs 86.7% survival in MN9D/Bax). Similar patterns were observed in two other MN9D/Bax cell lines. Cleavage of poly(ADP-ribose)polymerase caused by nigericin was greatly attenuated in MN9D/Bax cells suggesting that, like Bcl-2, Bax suppresses nigericin-induced cell death by inhibiting the activation of cysteine proteases. Thus, our data imply that Bax acts as a negative or positive regulator of cell death depending on the type of death stimulus applied to the cell.

Animals↗

PubMed1997

The apoptosis and proliferation of SAC-activated B cells by IL-10 are associated with changes in Bcl-2, Bcl-xL, and Mcl-1 expression.

Interleukin-10 (IL-10), a cytokine from mouse Th2 cells and macrophage that inhibits IL-2 and IFN-gamma production by Th1 cells, has been reported to stimulate growth and differentiation of B cells activated by CD40 or antigen receptor crosslinking. Our early observation revealed that IL-10 had B cell growth factor (BCGF) activity in human B cells preactivated with SAC or anti-Ig. The responsiveness of the preactivated B cells to IL-10 greatly increased when B cells were activated in the presence of IL-2, whereas IL-10 has no BCGF activity when added at the initiation of activation by SAC. To investigate the dual effects (proliferation and apoptosis) of IL-10 on B cells, the expression of a panel of bcl-2 protoncogene family members, bcl-2, bcl-x, mcl-1, and bax, was analyzed when B cells were activated by SAC. Bcl-xL protein was not expressed in the small resting B cells but was induced by SAC stimulation, reaching its peak at 48 hr. The addition of IL-2 further augmented the Bcl-xL expression with the same kinetics, whereas Bcl-2 and Mcl-1 were expressed by resting B cells and enhanced by SAC stimulation. However, the addition of IL-10 at the initiation of activation down-regulated Bcl-xL, Bcl-2, and Mcl-1 expression. At the same time, B cell proliferation was inhibited and apoptotic cell number increased, suggesting the growth arrest and/or apoptosis of B cells. The apoptosis of SAC-activated B cells by IL-10 was further confirmed by propidium iodide-staining and Annexin V-FITC-staining methods. In contrast, IL-10 failed to down-regulate the Bcl-xL and Bcl-2 expression but rather augmented the expression of Mcl-1 of B cells after preactivation for 48 hr with SAC and IL-2. Under this culture condition, B cells responded to IL-10 to proliferate and differentiate, while IL-2 and IL-10 had an additive or synergistic effect. Taken together, our data suggest that IL-10 acts on the induction stage of Bcl-xL expression and regulates the apoptosis and proliferation of SAC-activated B cells through their bcl-2 family gene expression.

Animals↗

PubMed1997

Immunolocalization of the ICE/Ced-3-family protease, CPP32 (Caspase-3), in non-Hodgkin's lymphomas, chronic lymphocytic leukemias, and reactive lymph nodes.

Immunohistochemical analysis of the apoptosis-effector protease CPP32 (Caspase-3) in normal lymph nodes, tonsils, and nodes affected with reactive hyperplasia (n = 22) showed strong immunoreactivity in the apoptosis-prone germinal center B-lymphocytes of secondary follicles, but little or no reactivity in the surrounding long-lived mantle zone lymphocytes. Immunoblot analysis of fluorescence-activated cell sorted germinal center and mantle zone B cells supported the immunohistochemical results. In 22 of 27 (81%) follicular small cleaved cell non-Hodgkin's B-cell lymphomas, the CPP32-immunopositive germinal center lymphocytes were replaced by CPP32-negative tumor cells. In contrast, the large cell component of follicular mixed cells (FMs) and follicular large cell lymphomas (FLCLs) was strongly CPP32 immunopositive in 12 of 17 (71%) and in 8 of 14 (57%) cases, respectively, whereas the residual small-cleaved cells were poorly stained for CPP32 in all FLCLs and in 12 of 17 (71%) FMs, suggesting that an upregulation of CPP32 immunoreactivity occurred during progression. Similarly, cytosolic immunostaining for CPP32 was present in 10 of 12 (83%) diffuse large cell lymphomas (DLCLs) and 2 of 3 diffuse mixed B-cell lymphomas (DMs). Immunopositivity for CPP32 was also found in the majority of other types of non-Hodgkin's lymphomas studied. Plasmacytomas were CPP32 immunonegative in 4 of 12 (33%) cases, in contrast to normal plasma cells, which uniformly contained intense CPP32 immunoreactivity, implying downregulation of CPP32 in a subset of these malignancies. All 12 peripheral blood B-cell chronic lymphocyte leukemia specimens examined were CPP32 immunopositive, whereas 3 of 3 small lymphocytic lymphomas were CPP32 negative, suggesting that CPP32 expression may vary depending on the tissue compartment in which these neoplastic B cells reside. The results show dynamic regulation of CPP32 expression in normal and malignant lymphocytes.

Animals↗

PubMed1997

Dissociation between cell cycle arrest and apoptosis can occur in Li-Fraumeni cells heterozygous for p53 gene mutations.

The radiation response was investigated in two lymphoblastoid cell lines (LBC) derived from families with heterozygous germ-line missense mutations of p53 at codon 282 (LBC282) and 286 (LBC286), and compared to cells with wt/wt p53(LBC-N). By gel retardation assays, we show that p53-containing nuclear extracts from irradiated LBC282 and LBC286 markedly differ in their ability to bind to a p53 DNA consensus sequence, the former generating a shifted band whose intensity is 30-40% that of LBC-N, the latter generating an almost undetectable band. Unlike LBC286, which fail to arrest in G1 after irradiation, LBC282 have an apparently normal G1/S checkpoint, as they arrest in G1, like LBC-N. While in LBC-N, accumulation of p53 and transactivation of p21WAF1 increase rapidly and markedly by 3 h after exposure to gamma-radiation, in LBC286 there is only a modest accumulation of p53 and a significantly delayed and quantitatively reduced transactivation of p21WAF1. Instead, in LBC282 while p53 levels rise little after irradiation, p21WAF1 levels increase rapidly and significantly as in normal LBC. Apoptotic cells present 48 h after irradiation account for 32% in LBC-N, 8-9% in LBC282 and 5-7% in LBC286, while the dose of gamma-radiation required for killing 50% of cells (LD50) is 400 rads, 1190 rads and 3190 rads, respectively, hence indicating that the heterozygous mutations of p53 at codon 282 affects radioresistance and survival, but not the G1/S cell cycle control. In all LBC tested, radiation-induced apoptosis occurs in all phases of the cell cycle and appears not to directly involve changes in the levels of the apoptosis-associated proteins bcl-2, bax and mcl-1. Both basal as well as radiation-induced p53 and p21WAF1 proteins are detected by Western blotting of FACS-purified G1, S and G2/M fractions from the three cell lines. p34CDC2-Tyr15, the inactive form of p34CDC2 kinase phosphorylated on Tyr15, is found in S and G2/M fractions, but not in G1. However, 24 h after irradiation, its levels in these fractions diminish appreciably in LBC-N but not in the radioresistant LBC286 and LBC282. Concomitantly, p34CDC2 histone H1 kinase activity increases in the former, but not in the latter cell lines, hence suggesting a role for this protein in radiation-induced cell death. Altogether, this study shows that, in cells harbouring heterozygous mutations of p53, the G1 checkpoint is not necessarily disrupted, and this may be related to the endogenous p53 heterocomplexes having lost or not the capacity to bind DNA (and therefore transactivate target genes). Radiation-induced cell death is not cell cycle phase specific, does not involve the regulation of bcl-2, bax or mcl-1, but is associated with changes in the phosphorylation state and activation of p34CDC2 kinase.

Apoptosis↗

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↗

PubMed1997

Immunohistochemical analysis of in vivo patterns of expression of CPP32 (Caspase-3), a cell death protease.

The in vivo patterns of CPP32 (Caspase-3) gene expression were determined using an immunohistochemical approach and paraffin-embedded normal human tissues. A rabbit polyclonal antiserum was generated against recombinant human CPP32 protein and shown to be specific by immunoblot analysis of various human tissues and cell lines. CPP32 immunoreactivity was selectively found in certain cell types and was typically present within the cytosol, although occasional cells also contained nuclear immunostaining. CPP32 immunostaining was easily detected, for example, in epidermal keratinocyes, cartilage chondrocytes, bone osteocytes, heart myocardiocytes, vascular smooth muscle cells, bronchial epithelium, hepatocytes, thymocytes, plasma cells, renal tubule epithelium, spermatogonia, prostatic secretory epithelial cells, uterine endometrium and myometrium, mammary ductal epithelial cells, and the gastrointestinal epithelium of the stomach, intestine, and colon. In contrast, little or no CPP32 immunoreactivity was observed in endothelial cells, alveolar pneumocytes, kidney glomeruli, mammary myoepithelial cells, Schwann cells, and most types of brain and spinal cord neurons. Consistent with a role for CPP32 in apoptotic cell death, clear differences in the relative intensity of CPP32 immunostaining were noted in some shorter-lived types of cells compared to longer-lived, including (a) germinal center (high) versus mantle zone (low) B lymphocytes within the secondary follicles of lymph nodes, spleen, and tonsils; (b) mature neutrophils (high) versus myeloid progenitor cells (low) in bone marrow; (c) corpus luteal cells (high) versus follicular granulosa cells (low) in the ovary; and (d) prostate secretory epithelial cells (high) versus basal cells (low). These findings establish for the first time the cell type- and differentiation-specific patterns of expression of an interleukin-1beta converting enzyme/CED-3 (Caspase) family protease.

Animals↗

PubMed1997

Bcl-2 and Bax expression in thyroid tumours. An immunohistochemical and western blot analysis.

Bcl-2 and Bax proteins, which are involved in repressing and promoting programmed cell death, respectively, have been investigated immunohistochemically and by Western blot analysis in a series of thyroid tumours. Three immunostaining patterns were identified. Benign lesions and well-differentiated thyroid carcinomas displayed a profile similar to that of normal follicular epithelium, in which Bcl-2 immunostaining was predominant. Thyroid carcinomas associated with an aggressive behaviour, such as the tall-cell variant of papillary carcinoma and the poorly differentiated carcinomas, co-expressed both proteins. Finally, anaplastic carcinomas expressed only the Bax protein. Western blot analyses revealed that the anti-Bcl-2 antibody recognized two bands, of molecular weights 21 kDa and 25 kDa. This was only seen in the tall-cell papillary carcinomas and in the anaplastic carcinomas.

Adult↗

PubMed1997

Reversal of EBV immortalization precedes apoptosis in IL-6-induced human B cell terminal differentiation.

Cell death in B cell terminal differentiation rapidly follows cell cycle arrest in IL-6 differentiation of EBV-immortalized, IgG-bearing human lymphoblastoid cells in vitro. G1 arrest is now found to coincide with repression of EBNA2 and LMP1, two EBV genes essential for B cell transformation, without activation of the viral lytic cycle. IL-6-differentiated B cells die by apoptosis, as evidenced by increases in Annexin V binding activity, PARP cleavage, and chromatin disorganization. Expression of Mcl-1, a Bcl-2 family member, was specifically induced during IL-6 differentiation and down-regulated during apoptosis. Thus, IL-6 reverses EBV immortalization and activates the terminal differentiation program in IgG-bearing human B lymphoblastoid cells, including regulation of an anti-apoptotic gene to coordinate differentiation, cell cycle arrest, and cell death.

Animals↗

PubMed1997

Role of apoptosis and apoptosis-related proteins in the cisplatin-resistant phenotype of human tumor cell lines.

Since apoptosis is the primary mode of cell death induced by cisplatin, the role of apoptosis and apoptosis-related gene products in cisplatin resistance was investigated in four human cisplatin-resistant cell lines of different tumour type. A common feature of the resistant sublines was a reduced susceptibility to drug-induced apoptosis compared to parental sensitive lines. Loss of wild-type p53 function was not a general event associated with the development of drug resistance. An increased bcl-2 expression was found in resistant cells characterized by mutant p53 (A431/Pt and IGROV-1/Pt), whereas in osteosarcoma (U2-OS/Pt) and in ovarian carcinoma (A2780/CP) cells with wild-type p53, bcl-2 levels were markedly reduced. U2-OS/Pt cells had a 16-fold increase in the level of Bcl-xL protein. Stable transfection of U2-OS cells with bcl-xL cDNA conferred a low level of drug resistance to cisplatin, suggesting that overexpression of this gene contributes to the cisplatin-resistant phenotype of this osteosarcoma cell system. In conclusion, these observations suggest a variable contribution of apoptosis-related genes to cisplatin resistance depending on the biological background of the cell system and presumably reflecting different pathways of apoptosis.

Journal Article↗

PubMed1997

Spontaneous overexpression of the long form of the Bcl-X protein in a highly resistant P388 leukaemia.

A novel resistant variant of murine P388 leukaemia, P388/SPR, was identified by de novo resistance to doxorubicin (DOX) in vivo. This mutant displayed a similar level of cross-resistance to etoposide (VP-16) and other topoisomerase II (topo II) inhibitors. Further analysis of the phenotype revealed a broad cross-resistance to vinca alkaloids, alkylating agents, antimetabolites, aphidicolin and UV light. Low-level expression of mdr1 and P-glycoprotein (P-gp), as well as a modest impairment of cellular drug accumulation and partial reversion of resistance to DOX and VP-16 by cyclosporine, confirmed a moderate role of P-gp in conferring drug resistance in P388/SPR cells. Consistent changes in neither topo II expression or activity nor glutathione metabolism could be detected. Induction of apoptosis was significantly reduced in P388/SPR cells, as indicated by minimal DNA fragmentation. Analysis of oncogenes regulating apoptotic cell death revealed a marked decrease of bcl-2 in combination with a moderate reduction of bax protein, but a striking overexpression of the long form of the bcl-X protein. Transfection of human bcl-X-L into P388 cells conferred drug resistance similar to that of P388/SPR cells. The data suggest that overexpression of bcl-X-L results in an unusual phenotype with broad cross-resistance to non-MDR-related cytotoxins in vitro, and provide an interesting example of spontaneous overexpression of another member of the bcl-2 gene family in cancer.

ATP Binding Cassette Transporter, Subfamily B, Mem↗