Biomedical subjects

J C Reed

Publications and source records attributed to J C Reed.

At least 397 records · Page 22Linked to original sources

PubMed1995

Programmed cell death and expression of the protooncogene bcl-2 in myocytes during postnatal maturation of the heart.

To determine whether programmed myocyte cell death is a major component of the differential growth adaptation of the right and left ventricle during development, the formation of DNA strand breaks in myocyte nuclei was identified and quantitated in hearts of rats at the end of gestation and at 1, 5, 11, and 21 days after birth. Incorporation of BrdU in left and right ventricular myocytes was also evaluated. Moreover, the expression of bcl-2 in myocytes was determined. Programmed myocyte cell death was absent in the fetal heart but affected the myocardium postnatally. This phenomenon was no longer detectable at 21 days. DNA strand breaks in nonmyocyte nuclei were present at all time intervals. Quantitatively, 10.4, 6.1, and 2.5 myocyte nuclei/10,000 nuclei exhibited DNA degradation at 1 day in the right ventricle, interventricular septum, and left ventricule, respectively. Corresponding values at 5 days were 3.7, 3.5, and 2.0 myocyte nuclei/10,000 nuclei. At 11 days, programmed cell death involved 1.2, 1.5, and 0.53 myocyte nuclei/10,000 nuclei in these three regions of the heart. The 4.2-fold, 1.9-fold, and 2.3-fold greater magnitude of programmed cell death in the right ventricle at 1, 5, and 11 days was statistically significant. In contrast, BrdU incorporation in myocytes decreased in a comparable manner in the left and right ventricles with maturation. Importantly, bcl-2 mRNA levels were high in fetal myocytes, decreased markedly at 1 and 5 days, and progressively increased at 11 and 21 days. The expression of bcl-2 was lower in right than in left ventricular myocytes. In conclusion, programmed myocyte cell death is inversely related to bcl-2 expression and affects the right ventricle more than the left ventricle during postnatal development. This phenomenon may be crucial in the modulation of the number of myocytes in the two ventricles during the transition from the fetal to the adult circulatory system.

Aging↗

PubMed1995

Bcl-2 family proteins: regulators of chemoresistance in cancer.

Knowledge about the mechanisms that regulate the expression of the family of BCL-2 genes and the biochemical characteristics of their encoded proteins is beginning to provide new insights into the origins of cancer and our all too often inability to adequately treat it. With better understanding of the functional significance of these protein-protein interactions involving Bcl-2, Bax, and other members of the Bcl-2 protein family and with insights into the structural details of these interactions, it may eventually be possible to develop novel pharmacological agents that improve tumor responses to currently available anticancer drugs and that have other clinically relevant uses as well.

Animals↗

PubMed1995

R-Ras promotes apoptosis caused by growth factor deprivation via a Bcl-2 suppressible mechanism.

The Bcl-2 protein is an important regulator of programmed cell death, but the biochemical mechanism by which this protein prevents apoptosis remains enigmatic. Recently, Bcl-2 has been reported to physically interact with a member of the Ras superfamily of small GTPases, p23-R-Ras. To examine the functional significance of R-Ras for regulation of cell death pathways, the IL-3-dependent cells 32D.3 and FL5.12 were stably transfected with expression plasmids encoding an activated form (38 Glycine-->Valine) of R-Ras protein. R-Ras(38V)-producing 32D.3 and FL5.12 cells experienced increased rates of apoptotic cell death relative to control transfected cells when deprived of IL-3. Analysis of several independent clones of transfected 32D.3 cells revealed a correlation between higher levels of R-Ras protein and faster rates of cell death upon withdrawal of IL-3 from cultures. 32D.3 cells cotransfected with R-Ras(38V) and Bcl-2 exhibited prolonged cell survival in the absence of IL-3, equivalent to 32D.3 cells transfected with Bcl-2 expression plasmids alone. R-Ras(38V) also increased rates of cell death in serum-deprived NIH-3T3 cells, and Bcl-2 again abrogated most of this effect. The ratio of GTP and GDP bound to R-Ras(38V) was not significantly different in control 32D.3 cells vs those that overexpressed Bcl-2, indicating that Bcl-2 does not abrogate R-Ras-mediated effects on cell death by altering R-Ras GDP/GTP regulation. Moreover, purified Bcl-2 protein had no effect on the GTPase activity of recombinant wild-type R-Ras in vitro. When expressed in Sf9 cells using recombinant baculoviruses, R-Ras(38V) bound to and induced activation of Raf-1 kinase irrespective of whether Bcl-2 was coproduced in these cells, suggesting that Bcl-2 does not nullify R-Ras effects by interfering with R-Ras-mediated activation of Raf-1 kinase. Taken together, these findings suggest that R-Ras enhances the activity of a cell death pathway in growth factor-deprived cells and imply that Bcl-2 acts downstream of R-Ras to promote cell survival.

3T3 Cells↗

PubMed1995

Immunoblot analysis of cellular expression of Bcl-2 family proteins, Bcl-2, Bax, Bcl-X and Mcl-1, in human peripheral blood and lymphoid tissues.

The ability of Bcl-2 to inhibit apoptotic cell death is well established. Several homologues of the bcl-2 gene, such as bax, bcl-x or mcl-1, have recently been identified. Like Bcl-2, both Bcl-XL and Mcl-1 appear to function as repressors of apoptotic cell death, whereas Bax facilitates it, indicating possible interactions among them in the control of cellular survival. To investigate the in vivo role of expression of bcl-2 gene family products, immunoblot analysis using corresponding specific antisera was performed for peripheral blood cells and some lymphoid tissues in humans. We demonstrated that all Bcl-2 family proteins were expressed at various levels in hematolymphoid cell subpopulations isolated from peripheral blood, tonsil, spleen and thymus. Lymphoid expression of Bcl-2 family proteins tended to increase following activation, but declined with time in culture. Loss of Bcl-2 in cultured lymphoid cells was especially marked. Sole expression of Bax, but not other members of the Bcl-2 family, was observed on neutrophils, seemingly reflecting their shortest life-span among blood leukocytes. The results support the notion that a balance of expression of Bcl-2 family proteins may regulate the life and death of hematolymphoid cells at different stages of cell differentiation and activation.

Acute Disease↗

PubMed1995

Regulation of apoptosis by bcl-2 family proteins and its role in cancer and chemoresistance.

In essentially all tissues that have self-renewal capacity, there exists a delicate balance between cell production by mitogenesis and cell loss due to programmed cell death (PCD), which maintains total cell numbers within physiologically appropriate ranges. Genetic alterations that either dysregulate the cell division process, resulting in faster cell proliferation, or that affect physiological cell death mechanisms that cause slower rates of cell loss, occur frequently in human tumor cells and contribute to the clonal expansion of cancer cells in vivo. PCD is an active form of cell suicide that sometimes requires new gene expression for its initiation and that in many, but not all, cases culminates in a characteristic set of biochemical and morphological events. These include genomic DNA cleavage by endonucleases, chromatin condensation (pyknosis), nuclear fragmentation, proteolysis of cytoskeletal and other proteins, plasma membrane blebbing, and cell shrinkage. In many ways, these biochemical events can be viewed as a form of cellular autodigestion in which the macromolecular components of cells are degraded so that their constituent subunits can be recycled in the body. When present in their classical form, the morphological events accompanying this type of cell death are broadly termed apoptosis. Though well known for its role in the normal physiological cell death mechanisms that maintain tissue homeostasis, a wide variety of pathological conditions and external factors can trigger the PCD pathway. Included among these apoptotic stimuli are essentially all chemotherapeutic drugs and radiation, a finding of considerable relevance to our understanding of how currently available treatments of cancer work and for devising strategies for improving them. In this review, the regulation of PCD by members of the bcl-2 family of proteins is discussed, primarily within the context of human cancers where abnormalities in the expression of BCL2 family genes frequently occur and contribute both to the origins of cancer and our difficulty in treating it.

Animals↗

PubMed1995

Mediastinal staging of non-small-cell lung cancer with positron emission tomography.

To determine the usefulness of positron emission tomography with fluoro-2-deoxyglucose (PET-FDG) in assessing mediastinal disease in patients with non-small-cell lung cancer (NSCLC) and to compare its yield to that of computed tomography (CT), we performed a prospective consecutive sample investigation in a university hospital and its related clinics. In 30 patients with NSCLC with clinical stage I (T1-2, NO, MO) disease, we compared the results of chest CT and PET-FDG with the findings at surgical exploration of the mediastinum. Seven (77%) of nine patients with surgically proven mediastinal metastasis were identified by the PET-FDG results, with four false-positives in 21 patients with negative lymph node dissections (p = 0.004). Using the results of pathologic examination of mediastinal lymph nodes as the criterion standard, the diagnostic sensitivity, specificity, accuracy, positive predictive value (PPV), and negative predictive value (NPV) for PET-FDG imaging of mediastinal metastases were 78%, 81%, 80%, 64%, and 89%, respectively. The sensitivity, specificity, accuracy, PPV, and NPV for chest CT in the detection of mediastinal metastasis were 56%, 86%, 77%, 63%, and 87%, respectively. CT and PET-FDG results agreed in 21 patients. The diagnostic accuracy of the combined imaging modalities was 90%. We concluded that mediastinal uptake of FDG correlates with the extent of mediastinal involvement of NSCLC and may contribute to preoperative staging. PET-FDG imaging complements chest CT in the noninvasive evaluation of NSCLC, and strategies for its use merit further investigation.

Aged↗

PubMed1995

Protooncogene bcl-2 gene transfer abrogates Fas/APO-1 antibody-mediated apoptosis of human malignant glioma cells and confers resistance to chemotherapeutic drugs and therapeutic irradiation.

The majority of human malignant glioma cells express Fas/APO-1 and are susceptible to Fas/APO-1 antibody-mediated apoptosis in vitro. The sensitivity of Fas/APO-1-positive glioma cell lines to Fas/APO-1 antibody-mediated killing correlates inversely with the constitutive expression of the antiapoptotic protooncogene bcl-2. Here we report that BCL-2 protein expression of human glial tumors in vivo correlates with malignant transformation in that BCL-2 immunoreactive glioma cells were more abundant in WHO grade III/IV gliomas than in grade I/II gliomas. Fas/APO-1 antibody-sensitive human glioma cell lines stably transfected with a murine bcl-2 cDNA acquired resistance to Fas/APO-1 antibody-mediated apoptosis. Forced expression of bcl-2 also attenuated TNF alpha-mediated cytotoxicity of glioma cell lines in the presence of actinomycin D and cycloheximide and conferred partial protection from irradiation and the cancer chemotherapy drugs, cisplatin and BCNU. Preexposure of the glioma cell lines to the cytokines, IFN gamma and TNF alpha, which sensitize for Fas/APO-1-dependent killing, partially overcame bcl-2-mediated rescue from apoptosis, suggesting that multimodality immunotherapy involving cytokines and Fas/APO-1 targeting might eventually provide a promising approach to the treatment of human malignant gliomas.

Animals↗

PubMed1995

Inhibition of ceramide-induced apoptosis by Bcl-2.

Ceramide, a long chain sphingolipid that is generated intracellularly upon hydrolysis of membrane-associated sphingomyelin, has recently been implicated as a second messenger-like molecule that is produced distal to ligation of the tumour necrosis factor receptor type 1 (TNFR1), as well as the related Fas (CD95/Apo-1) molecule. It is well established that ligation of TNFR1 or Fas leads to apoptosis in most cases. Furthermore, it has also recently been demonstrated that exposure to cell-permeable synthetic ceramides can result in apoptosis in many cases. These and other observations have led to the hypothesis that accumulation of intracellular ceramide may be a common element of several pathways that result in apoptosis. Here we show that exposure to synthetic ceramides triggers apoptosis in the human T lymphoblastoid cell lines, CEM and Jurkat, and that overexpression of the apoptosis-repressor protein, Bcl-2, renders these cells resistant to the apoptosis-inducing effects of ceramide, as well as to several other stimuli. Since exposure to ceramides can result in either cell proliferation, differentiation, cycle arrest, or death, the level of Bcl-2 expression in a cell may be an important factor in determining the outcome of signals that result in intracellular generation of this sphingolipid.

Journal Article↗

PubMed1995

Biochemical and functional comparisons of Mcl-1 and Bcl-2 proteins: evidence for a novel mechanism of regulating Bcl-2 family protein function.

Mcl-1 is a recently described homologue of Bcl-2 whose function and biochemical characteristics remain poorly defined. Gene transfer experiments in lnterleukin-3 (IL-3)-dependent myeloid progenitor 32D.3 cells and pro-B-lymphoid FL5.12 cells demonstrated that enforced production of high levels of Mcl-1 protein failed to prolong the survival of cells when cultured in the absence of IL-3, whereas Bcl-2 did delay cell death. Mcl-1 also did not prolong the survival in vitro of 32D.3 cells that had been induced to differentiate into mature neutrophils using Granulocyte-Colony Stimulating Factor (G-CSF), whereas Bcl-2 did. 32D.3 and FL5.12 cells co-transfected with Mcl-1 and Bcl-2 displayed survival kinetics essentially identical to cells transfected with Bcl-2 alone, when cultured in the absence of IL-3, indicating that Mcl-1 neither enhances nor impairs Bcl-2 function. In contrast to the lack of effects of Mcl-1 in 32D.3 and FL5.12 cells, Mcl-1 (like Bcl-2) was able to neutralise Bax-induced cytotoxicity in yeast (S. cerevisiae). Moreover, the recombinant GST-Mcl-1 protein bound specifically to in vitro translated Bax protein, as well as to Bax protein present in detergent lysates prepared from 32D.3 and FL5.12 cells, based on in vitro binding assays. However, Mcl-1 and Bax proteins could not be co-immunoprecipitated from control and transfected 32D.3 and FL5.12 cells, whereas Bcl-2 and Bax were easily co-immunoprecipitated under the same conditions. The findings suggest that while Mcl-1 has the capacity to bind to and neutralise the cell death promoting activity of Bax, other factors such as perhaps additional proteins or undefined post-translational modifications may influence its ability to bind to Bax in vivo and thus affect its function as a cell death blocker.

Journal Article↗

PubMed1995

Upregulation of bax protein levels in neurons following cerebral ischemia.

The patterns of expression of the bcl-2, bax, and bci-X genes were examined immunohistochemically in neurons of the adult rat brain before and after 10 min of global ischemia induced by transient cardiac arrest. High levels of the cell death promoting protein Bax and concomitant low levels of the apoptosis-blocking protein Bcl-2 were found in some populations of neurons that are particularly sensitive to cell death induced by transient global ischemia, such as the CA1 sector of the hippocampus and the Purkinje cells of the cerebellum. Moreover, within 0.5 to 3 hr after an ischemic episode, immunostaining for Bax was markedly increased within neurons with morphological features of degeneration in many regions of the brain. Use of a two-color staining method for simultaneous analysis of Bax protein and in situ detection of DNA-strand breaks revealed high levels of Bax immunoreactivity in many neurons undergoing apoptosis. Postischemic elevations in Bax protein levels in the hippocampus, cortex, and cerebellum were also demonstrated by immunoblotting. At early times after transient ischemia, regulation of Bcl-2 and Bcl-x protein levels varied among neuronal subpopulations, but from 3 hr on, those neurons with morphological evidence of degeneration uniformly contained reduced levels of Bci-2 and particularly Bci-X immunoreactivity. The findings suggest that differential expression of some members of the bcl-2 gene family may play an important role in determining the relative sensitivity of neuronal subpopulations to ischemia and that postischemic alterations in the expression of bax, bcl-2, and bcl-x may contribute to the delayed neuronal cell death that occurs during the repurfusion phase after a transient ischemic episode.

Animals↗

PubMed1995

Immunohistochemical analysis of Bcl-2 protein regulation in cutaneous melanoma.

Cutaneous melanoma is becoming increasingly common. Genetic and environmental factors are thought to play a role in its pathogenesis. We have previously shown that normal human melanocytes strongly express the oncoprotein, Bcl-2. To determine the role of Bcl-2 in melanocytic tumors, we studied human benign nevi and melanomas for expression of Bcl-2 protein using immunohistochemistry. Our results show that benign melanocytes from 3 of 4 normal skin biopsies and 5 of 7 common acquired nevi strongly express Bcl-2. Conversely, only 3 of 23 primary cutaneous melanomas and 3 of 9 metastatic melanomas showed strong staining in comparison with melanocytes from normal skin and common acquired nevi (chi 2, P = 0.0021). Interestingly, 0 of 6 dysplastic nevi, a precursor of melanoma, demonstrated strong staining as compared with melanocytes and nevi (8 of 11; chi 2, P = 0.02), but similar expression to that of melanoma (6 of 32; chi 2, P = 0.6). We conclude that Bcl-2 expression decreases in malignant melanoma and suggest that this may be related to the autonomous growth characteristics of malignant melanoma.

Humans↗

PubMed1995

Bcl-2: prevention of apoptosis as a mechanism of drug resistance.

Programmed cell death (also known as apoptosis) plays an essential role in tissue homeostasis, where it ensures that new cell production in the body is offset by a commensurate rate of cell loss. Defects in the genetic pathway that regulate the cell death process can figure prominently in the origins of cancer and also in problems with cancer treatment. Eventually, it may be possible to develop novel treatments for cancer that specifically seek to modulate the physiologic cell death pathway as opposed to nearly all currently available drugs, which are intended to interfere with some aspect of the cell division cycle.

Animals↗

PubMed1995

Apoptosis and Bcl-2 expression in cultured murine splenic T cells.

To elucidate the mechanism by which bcl-2 affects apoptosis in post-thymic T cells, we investigated the expression of Bcl-2 protein in primary cultures of splenic T cells and in the interleukin-2 (IL-2)-dependent T-cell line CTLL-2. The overall level of Bcl-2 was determined by immunoblotting, and the variability in Bcl-2 expression was determined by flow cytometry. For a few days after concanavalin A (Con A) plus IL-2 activation, the overall level of Bcl-2 in T cells remains unchanged, but it becomes more heterogeneous. By 5 days after activation, the expression returns to a more homogeneous distribution, but it is increased up to threefold above pre-activation levels, depending upon the dose of IL-2 supplied. When Con A blasts or CTLL-2 cells are deprived of IL-2 for 24 hr, there is no change in their overall Bcl-2 levels which remain homogeneous even though almost half of the cells are apoptotic. However, when bcl-2 transfected CTLL-2 cells are deprived of IL-2, they do not undergo apoptosis, and their endogenous Bcl-2 protein level slowly decreases relative to their total protein. These data document the IL-2-dependent expression of Bcl-2 in activated T cells, confirm the ability of deregulated bcl-2 to inhibit the onset of apoptosis after IL-2 withdrawal, but suggest that, after IL-2 withdrawal, a drop in Bcl-2 levels relative to total protein levels does not precede apoptosis.

Animals↗

PubMed1995

Immunohistochemical analysis of Mcl-1 protein in human tissues. Differential regulation of Mcl-1 and Bcl-2 protein production suggests a unique role for Mcl-1 in control of programmed cell death in vivo.

The mcl-1 gene encodes an approximately 37-kd protein that has significant homology with Bcl-2, an inhibitor of programmed cell death that is expressed in many types of long-lived cells. In this study we determined the in vivo patterns of Mcl-1 protein production in normal human tissues by immunohistochemical means, using specific polyclonal antisera, and made comparisons with Bcl-2. Like Bcl-2, Mcl-1 immunostaining was observed in epithelial cells in a variety of tissues, including prostate, breast, endometrium, epidermis, stomach, intestine, colon, and respiratory tract. However, often the expression of mcl-1 and bcl-2 in complex epithelia occurred in gradients with opposing directions, such that Bcl-2 immunostaining tended to be higher in the less differentiated cells lining the basement membrane, whereas Mcl-1 immunostaining was more intense in the differentiated cells located in the upper layers of these epithelia. The in vivo patterns of mcl-1 and bcl-2 expression were also strikingly different in several other tissues as well. Within the secondary follicles of lymph nodes and tonsils, for example, germinal center lymphocytes were Mcl-1 positive but mostly lacked Bcl-2; whereas mantle zone lymphocytes expressed bcl-2 but not mcl-1. Intense Mcl-1 immunoreactivity was also detected in several types of neuroendocrine cells, including the adrenal cortical cells that are Bcl-2 negative, sympathetic neurons that also contain Bcl-2, a subpopulation of cells in the pancreatic islets, Leydig cells of the testis, and granulosa lutein cells of the ovarian corpus luteum but not in thyroid epithelium, which is strongly Bcl-2 positive. Little or no Mcl-1 was detected in neurons in the brain and spinal cord, in contrast to Bcl-2, which is present in several types of central nervous system neurons. Conversely, strong Mcl-1 immunostaining was found in cardiac and skeletal muscle, which contain comparatively less Bcl-2. Additional types of cells that are Bcl-2-negative but that expressed mcl-1 include chondrocytes and hepatocytes. These findings demonstrate that mcl-1 expression is widespread in vivo and imply that the Mcl-1 and Bcl-2 proteins fulfill different roles in the overall physiology of cell death regulation.

Apoptosis↗

PubMed1995

Immunohistochemical analysis of the Bcl-2 oncoprotein in human neuroblastomas. Comparisons with tumor cell differentiation and N-Myc protein.

BACKGROUND: The bcl-2 gene encodes a 26-kilodalton integral membrane oncoprotein that is noteworthy for its function as a blocker of programmed cell death, and ability to render cells resistant to killing by chemotherapeutic drugs and x-irradiation. EXPERIMENTAL DESIGN: To determine the in vivo patterns of bcl-2 expression in neuroblastomas (NBs), tumor specimens derived from 17 children with NB or ganglioneuromas were immunohistochemically evaluated using formalin-fixed, paraffin-embedded material and antibodies specific for the Bcl-2 and N-Myc proteins, or various markers typically used to assess the differentiation status of these tumors, including neuron-specific enolase, S-100 protein, beta 2-microglobulin (beta 2M) and the intermediate filament proteins, vimentin, and neurofilament light- and medium-chains. RESULTS: Using two-color immunohistochemical methods, Bcl-2 protein was found exclusively in tumor cells that did not contain N-Myc, an oncoprotein previously associated with poor prognosis in NB. Levels of Bcl-2 immunostaining were heterogeneous in the undifferentiated small, round cells typically seen in tumors with aggressive histology, and ranged from essentially undetectable, to strong in intensity. Somewhat higher levels of Bcl-2 immunostaining were found in the slightly larger, more differentiated neuroblastic cells that had more generous cytoplasm in these neoplasms. In contrast to the heterogeneous levels of Bcl-2 seen in undifferentiated NBs, Bcl-2 immunoreactivity was uniformly present at high levels in tumor cells that were more differentiated and judged to be similar to immature ganglion cells at an intermediate stage of neuronal differentiation, based on morphologic characteristics and immunophenotyping with antibodies specific for various differentiation markers. The still more differentiated ganglionic cells seen in ganglioneuroblastomas and ganglioneuromas exhibited much less intense immunoreactivity with anti-Bcl-2 antibodies, suggesting down-regulation of bcl-2 during final terminal differentiation. In contrast to cells with neuronal features, the stromal Schwann cells were uniformly negative for Bcl-2 protein in all histologic grades of tumors. Thus, bcl-2 expression in NBs was limited to cells of the neuronal lineage and tended to be highest in tumor cells with the characteristics of ganglionic cell precursors. The expression of bcl-2 in the normal fetal and postnatal adrenal medulla and sympathetic ganglia paralleled that seen in NBs, consistent with previous suggestions that NB reflects a block in the normal differentiation process. CONCLUSIONS: Taken together, these findings indicate that the levels of the Bcl-2 protein are developmentally regulated in normal and neoplastic cells of the sympathetic branch of the autonomic nervous system, and suggest that this oncoprotein may be useful as a differentiation marker for subclassification of NBs for prognostic purposes and for investigations of normal neuronal maturation. Furthermore, strong Bcl-2 immunoreactivity was detected in islets of residual tumor cells in 6 of 7 specimens obtained from 5 patients after therapy, suggesting that this oncoprotein may be cytoprotective for NB cells in vivo.

Adolescent↗

PubMed1995

The bcl-2 proto-oncogene and the gastrointestinal epithelial tumor progression model.

We report overexpression of the proto-oncogene bcl-2 in gastrointestinal adenocarcinoma and its precursor lesions. The bcl-2 proto-oncogene is centrally involved in the oncogenesis of human follicular lymphoma via a chromosomal translocation t(14;18)(q32;q21) and is also expressed in the epithelial regenerative compartment or the basal crypts of the normal colon and small intestine. We describe an immunohistochemical analysis of fixed, paraffin-embedded tissue using both a polyclonal rabbit and a monoclonal mouse antibody to the Bcl-2 protein. In addition to confirming bcl-2 expression in normal colonic and small intestinal crypts, we also observed expression in the gastric epithelial regenerative compartment, the mucous neck region. No increased expression was found in nonneoplastic or inflammatory gastrointestinal conditions, including ulcerative colitis, Crohn's disease, or inflammatory or hamartomatous polyps. Increased bcl-2 expression, however, was present in hyperplastic colonic polyps and in the majority of dysplastic lesions, from the earliest precursors through large adenomas, high grade flat dysplasia, and adenocarcinoma, all in comparison with adjacent internal control normal epithelium. Increased expression was present in dysplastic glandular lesions from all gastrointestinal sites, including colon, small bowel, and stomach. Furthermore, bcl-2 expression was frequently abnormal in nondysplastic epithelium surrounding dysplastic lesions, suggesting that altered expression occurred before the development of morphological dysplasia. Specifically, directly contiguous morphologically nondysplastic epithelium often showed abnormal bcl-2 expression throughout the full length of the crypt-villus axis. This expression pattern gradually diminished to involve only the crypt base (the normal pattern of expression), proceeding away from malignant or dysplastic lesions. Abnormal bcl-2 immunoreactivity in 1), the earliest precursor dysplastic lesions and its persistence throughout neoplastic progression and 2), contiguous morphologically unaltered nondysplastic epithelium suggests that bcl-2 alterations occur early during the morphological and molecular sequence of events leading to gastrointestinal epithelial neoplasia.

Adenocarcinoma↗

PubMed1995

Effects of modulators of protein kinases on taxol-induced apoptosis of human leukemic cells possessing disparate levels of p26BCL-2 protein.

Taxol-induced polymerization of tubulin into stable microtubules and cell cycle metaphase arrest have been demonstrated to result in internucleosomal DNA fragmentation and morphological features of apoptosis in human leukemia cells. Recent studies have also shown that Taxol-induced apoptosis, but not Taxol-induced microtubular bundling or mitotic arrest, is significantly inhibited in cells that overexpress the bcl-2 gene product p26BCL-2. In the present studies we examined the effects of several modulators of activities of protein kinases on Taxol-induced DNA fragmentation and apoptosis in human pre-B leukemia 697 cells transfected with the cDNA of the bcl-2 gene and expressing high intracellular levels of p26BCL-2 (697/BCL-2 cells). Treatment with 0.1-1.0 microM MTaxol for 24 h produced prolonged mitotic arrest of control 697/neo cells, which had been transfected with the neomycin resistance gene. This resulted in apoptosis-associated large DNA fragments ranging between 5 and 200 kb and internucleosomal DNA fragmentation. Cotreatment with the phorbol ester phorbol dibutyrate (PdBU) significantly reduced Taxol-induced internucleosomal and large DNA fragmentation and inhibited apoptosis of 697/neo cells. In contrast, a combined exposure to Taxol and staurosporine (ST; 5 or 50 ng/ml), a potent inhibitor of protein kinase C and other kinases, significantly increased DNA fragmentation and apoptosis of 697/neo cells. Additionally, in 697/BCL-2 cells, ST partially overcame the suppressive effects of high p26BCL-2 levels on Taxol-induced apoptosis. Cotreatment with the tyrosine kinase inhibitor Genistein (30 microM) markedly inhibited Taxol-induced DNA fragmentation and apoptosis of 697/neo cells. However, it is noteworthy that the modulations of Taxol-induced DNA fragmentation and apoptosis by PdBU, ST, and Genistein occurred without significant effects on Taxol-mediated mitotic arrest of 697/neo cells. These agents also did not affect intracellular p26BCL-2 levels in 697/neo or 697/BCL-2 cells. These findings indicate that Taxol-induced apoptosis can be modulated by agents that affect the activities of protein kinases, and these effects are not mediated by modulations of Taxol-induced mitotic arrest or by alterations of intracellular p26BCL-2 levels.

Antineoplastic Agents, Phytogenic↗

PubMed1994

Regulation of neuronal Bcl2 protein expression and calcium homeostasis by transforming growth factor type beta confers wide-ranging protection on rat hippocampal neurons.

Excessive activation of glutamate receptors accompanied by Ca2+ overloading is thought to be responsible for the death of neurons in various conditions including stroke and epilepsy. Neurons also die if deprived of important growth factors and trophic influences, conditions sensitive to certain oncogene products such as the Bcl2 protein. We now demonstrate that transforming growth factor type beta (TGF-beta) prevents neuronal Ca2+ overloading of rat hippocampal neurons in response to the glutamatergic agonist N-methyl-D-aspartate or the Ca2+ ionophore 4-Br-A23187 and, in addition, leads to a substantial increase in neuronal Bcl2 protein expression. Parallel cytotoxicity experiments demonstrate that treatment with TGF-beta protects rat hippocampal neurons from death induced by excitotoxicity, trophic factor removal, and oxidative injury. Thus, TGF-beta may protect against a wide range of toxic insults by regulating two factors with great importance for neuronal viability.

Animals↗