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G C Gobé

Publications and source records attributed to G C Gobé.

At least 19 recordsLinked to original sources
PubMed2001

Bcl-X(L) translocation in renal tubular epithelial cells in vitro protects distal cells from oxidative stress.

BACKGROUND: The molecular pathogenesis of different sensitivities of the renal proximal and distal tubular cell populations to ischemic injury, including ischemia-reperfusion (IR)-induced oxidative stress, is not well-defined. An in vitro model of oxidative stress was used to compare the survival of distal [Madin-Darby canine kidney (MDCK)] and proximal [human kidney-2 (HK-2)] renal tubular epithelial cells, and to analyze for links between induced cell death and expression and localization of selected members of the Bcl-2 gene family (anti-apoptotic Bcl-2 and Bcl-X(L), pro-apoptotic Bax and Bad). METHODS: Cells were treated with 1 mmol/L hydrogen peroxide (H2O2) or were grown in control medium for 24 hours. Cell death (apoptosis) was quantitated using defined morphological criteria. DNA gel electrophoresis was used for biochemical identification. Protein expression levels and cellular localization of the selected Bcl-2 family proteins were analyzed (Western immunoblots, densitometry, immunoelectron microscopy). RESULTS: Apoptosis was minimal in control cultures and was greatest in treated proximal cell cultures (16.93 +/- 4.18% apoptosis) compared with treated distal cell cultures (2.28 +/- 0.85% apoptosis, P < 0.001). Endogenous expression of Bcl-X(L) and Bax, but not Bcl-2 or Bad, was identified in control distal cells. Bcl-X(L) and Bax had nonsignificant increases (P> 0.05) in these cells. Bcl-2, Bax, and Bcl-X(L), but not Bad, were endogenously expressed in control proximal cells. Bcl-X(L) was significantly decreased in treated proximal cultures (P < 0.05), with Bax and Bcl-2 having nonsignificant increases (P> 0.05). Immunoelectron microscopy localization indicated that control and treated but surviving proximal cells had similar cytosolic and membrane localization of the Bcl-2 proteins. In comparison, surviving cells in the treated distal cultures showed translocation of Bcl-X(L) from cytosol to the mitochondria after treatment with H2O2, a result that was confirmed using cell fractionation and analysis of Bcl-X(L) expression levels of the membrane and cytosol proteins. Bax remained distributed evenly throughout the surviving distal cells, without particular attachment to any cellular organelle. CONCLUSION: The results indicate that in this in vitro model, the increased survival of distal compared with proximal tubular cells after oxidative stress is best explained by the decreased expression of anti-apoptotic Bcl-X(L) in proximal cells, as well as translocation of Bcl-X(L) protein to mitochondria within the surviving distal cells.

Animals↗
PubMed1999

Radiation-induced apoptosis and gene expression in neonatal kidney and testis with and without protein synthesis inhibition.

PURPOSE: To analyse the incidence of radiation-induced apoptosis, expression of two apoptosis-related genes, Bcl-2 and p53, and post-radiation levels of cell proliferation in the neonatal rat (4-5 days old) kidney and testis. MATERIALS AND METHODS: Apoptosis was quantified in control or treated kidney or testis at 2, 4, 6, 8 and 24h after 5 Gy of whole body X-irradiation (n=4 per group). Morphology (light and electron microscopy) and DNA gel electrophoresis were used to assess apoptosis. Temporal and spatial expression of Bcl-2 or p53 were analysed using immunohistochemistry. Administration of cycloheximide (1.5mg/kg) was used to determine whether new protein synthesis had a role in induction of apoptosis. Tritiated thymidine uptake and autoradiography were used to indicate alterations in cell proliferation (radiolabel administered 1 h prior to tissue collection) or S-phase cells undergoing radiation-induced apoptosis (radiolabel administered 1 h prior to irradiation). RESULTS: Apoptosis peaked at 4 h in the testis and 6 h in the kidney and was significantly higher in the renal nephrogenic zone than in the testis (p<0.05). Mitosis was almost completely negated after irradiation in both tissues. A higher proportion (almost fivefold) of the apoptotic cells died in S phase in the kidney than in the testis. Cycloheximide negated induction of apoptosis in the kidney, and markedly decreased apoptosis in the testis. Bcl-2 expression was highest in the differentiated zone of control kidneys and increased after irradiation in the nephrogenic zone, particularly near foci of apoptosis in developing nephrons. In the control testis, Sertoli cells had moderate expression of Bcl-2. After irradiation, there was complete absence of Bcl-2 expression in apoptotic Sertoli cells, with surviving cells increasing Bcl-2 expression. Irradiated kidney had more intense nuclear p53 expression compared with controls. In the testis, p53 that was present in controls continued to be expressed in surviving cells but not apoptotic cells in radiation-treated animals. CONCLUSIONS: Unique differences can be identified between the incidence and biomolecular control of radiation-induced apoptosis in the normal neonatal kidney and testis. These results may find application for minimizing damage to these normal neonatal tissues in the development of, for example, cancer treatment regimens.

Animals↗
PubMed1997

Expression and localization of the retinoblastoma gene during radiation-induced apoptosis in neonatal rat kidney.

An in vivo neonatal rat kidney model was used to study an association between expression and localization of the retinoblastoma tumor-suppressor gene (Rb), or its protein product (pRb), and localization of radiation-induced apoptosis. The rat kidney has two distinct zones of differentiation at birth-an outer nephrogenic zone, in which cells are undifferentiated and new nephrons are forming, and a differentiated zone internal to this zone that has essentially the adult kidney form. At 6 h after radiation (5 Gy), high levels of relatively synchronous apoptosis are induced in the nephrogenic zone, with little effect on the differentiated zone, and proliferation in the nephrogenic zone is almost totally inhibited by radiation treatment, again with little effect in the differentiated area. We have used our knowledge of this model to analyze control (sham-treated) and irradiated renal tissue for Rb mRNA transcript levels and localization (Northern blot and in situ hybridization (ISH)), pRb expression (Western blot and immunolocalization), apoptosis and mitosis (light and electron microscopy, and DNA gel electrophoresis for apoptosis), and cells in S-phase ([3H]thymidine uptake and autoradiography). Northern blots showed no detectable alteration in Rb transcript levels between control and irradiated tissues, whereas Western blots indicated increased expression of pRb in protein extracted from irradiated kidney compared with controls. ISH confirmed that Rb transcripts were not substantially altered in the nephrogenic and differentiated zones in control versus irradiated renal tissue. Immunolocalization of pRb demonstrated little effect in the differentiated zone, but in the nephrogenic zone pRb expression was increased, especially the S-shaped prenephrons, and was also found in many, but not all, apoptotic cells in this zone. The results link radiation-induced apoptosis and increased pRb expression in a zone of the neonatal kidney having a low level of cell differentiation.

Animals↗
PubMed1997

Analysis of apoptosis morphology in Epstein-Barr virus positive and negative Burkitt's lymphoma cells.

UNLABELLED: In recent studies of cycloheximide (CHX)-induced apoptosis in sublines of established Burkitt's lymphoma cell lines (BJA-B) both with and without Epstein-Barr virus (EBV) infection, we noticed two distinct types of apoptosis morphology. In the present paper, we have classified these, and further carried out a statistical analysis of their incidence in untreated and CHX-treated EBV-free (EBV(-)) and EBV-infected (EBV(+)) BJA-B cells. CLASSIFICATION: Both types of apoptosis morphology demonstrated typical nuclear and cytoplasmic condensation. However, "Type 1 apoptotic cells" (AP1) maintained a spherical or ovoid shape, but "Type 2 apoptotic cells" (AP2) were typified by the lobulation of their nuclear and cytoplasmic structures to form "clover leaf" shapes. Statistical analysis of incidence: The numbers of AP1 and AP2 cells were analysed using a chi 2 test, with results as follows: EBV(-) cells underwent AP1 in preference to AP2 (90.5% versus 9.5%) (p < 0.001), whilst EBV(+) cells had comparably more AP2, making AP1 and AP2 approximately equal (49.3% versus 50.7%) (p > 0.1). In EBV(-) cells, treatment with CHX had little effect on the ratios of differing apoptotic morphology. In contrast, in the EBV(+) cells, cell death was altered from AP2 (50.7%-->25.2%) towards AP1(49.3%-->74.8%) (p < 0.001). We propose that cellular proteins known to be associated with EBV infection not only protect the cells from apoptosis, but also affect the phenotype of apoptosis. This knowledge may be useful for defining possible mechanisms of apoptosis induction and/or inhibition in specific models.

Apoptosis↗
PubMed1996

Comparative alterations in p53 expression and apoptosis in the irradiated rat small and large intestine.

Temporal and spatial relationships between radiation-induced apoptosis and expression of p53 mRNA and protein were compared in rat small and large intestine. Apoptosis was quantified using morphological criteria, and p53 expression determined by immunohistochemistry or whole-tissue Northern analysis. In the small intestine, peak levels of apoptosis appeared earlier (4 h) than in the large intestine (6 h). p53 mRNA transcript levels in small and large intestine were not significantly altered from control levels at any time after treatment. However, in treated small and large intestine, cells showed increased positivity for p53 protein, increasing 10-fold over control levels 4-5 h after irradiation. A strong spatial relationship was found between high incidence apoptosis and p53 protein positivity. We compared published data of stem cell population positions for small and large intestine with our results. Target cells for apoptosis and p53 expression occurred at approximately fifth position from the crypt base of the small intestine, a zone coincident with stem cell population. Target cell position for apoptosis and p53 expression in the large intestine was again at fifth or sixth position from the base, but this zone is not the reported stem cell position (first or second position) for large intestine. Results from our model of radiation-induced intestinal apoptosis indicate that p53 protein is closely associated both temporally and spatially with the induction of apoptosis, and support the work of others in suggesting that p53 expression is modulated post-transcriptionally. Furthermore, our results support a hypothesis that apoptotic targeting of damaged stem cell populations, early response for apoptotic removal of DNA-damaged cells and/or early repair of these damage cells are all important parameters that determine differences in levels of tumorigenesis in the small and large intestine.

Animals↗
PubMed1996

Expression and localization of clusterin mRNA in the small and large intestine of the irradiated rat: its relationship with apoptosis.

Apoptotic elimination of intestinal cells following irradiation has been studied in the small and large intestine of the rat, and correlated with the level of expression and localization of clusterin mRNA. Clusterin was moderately expressed in normal intestine where only small levels of apoptosis were found. After irradiation, however, there was a temporal correlation between an increased apoptotic index and increased clusterin expression. Localization of clusterin mRNA by in situ hybridization identified extensive labelling in the lower part (Paneth cell region) of small intestinal crypt, whereas epithelial cells in the large intestine were diffusely labelled. Clusterin expression was not localized over apoptotic cells and its role may be as a cell protection factor for surviving cells, as had been suggested by others. Clusterin may also be involved in remodelling of the intestinal crypt after radiation damage, a process that includes altering cell-to-cell contact, apoptosis, and sloughing of the dead cells from the intestinal villi. Our results do show a close temporal link between apoptosis and clusterin expression, and, as such, expression of the gene may be a useful indicator of presence of apoptosis in the irradiated intestine.

Animals↗
PubMed1995

Cycloheximide-induced apoptosis in Burkitt lymphoma (BJA-B) cells with and without Epstein-Barr virus infection.

Epstein-Barr virus (EBV) has been shown, in many instances, to protect B cells from apoptosis via expression of select EBV proteins and up-regulation of bcl-2 or its homologues. However, at present little is known about the influence of EBV infection against cancer therapy-induced apoptosis in EBV-associated cancers. Many anti-cancer treatments act via inhibition of protein synthesis and so could influence the reported protein-dependent mechanisms involved in EBV inhibition of apoptosis. In the present study, Burkitt lymphoma (BJA-B) cells were treated with a potent protein synthesis inhibitor, cycloheximide (CHX). Two variants of BJA-B cells were used, one with EBV infection (EBV(+)), and one free of infection (EBV(-)). Cells were collected 0,3,6,12, 24 and 48 h after addition of either 1 or 50 micrograms/mL of CHX. Control cultures were untreated. Apoptosis was quantified using established morphological and biochemical characteristics, and protein concentrations assessed. CHX treatment of EBV(-) BJA-B cells induced massive levels of apoptosis. Apoptosis was inhibited, but remained significantly higher than that found in control cultures, in similarly treated EBV(+) cells. The study demonstrates that induction of apoptosis in EBV(-) and EBV(+) cells is not dependent on new protein synthesis and so may be indicative of a bcl-2 independent mechanism in this instance. The results have important implications for devising and assessing treatment of EBV-associated malignancies.

Antimetabolites↗
PubMed1995

Clusterin expression and apoptosis in tissue remodeling associated with renal regeneration.

To analyze the role of clusterin in renal diseases involving a regenerative process, we have used a novel rodent model to compare temporal and spatial expression of clusterin mRNA. Thus, renal artery stenosis was used to induce unilateral non-infarctive renal atrophy. After several weeks, when cellular pathology of atrophic kidneys involved minimal apoptosis or inflammatory response and mitosis was at normal levels, regeneration of atrophic kidneys was stimulated by removal of the contralateral healthy kidneys. The regrowth response was very rapid and involved renal hyperplasia rather than hypertrophy. Regenerating kidneys were studied 0, 4, 8, 24 hours and 2, 3, 5, 7, and 14 days after contralateral nephrectomy. Several parameters were compared: level and localization of clusterin mRNA; cell proliferation; cell dedifferentiation and redifferentiation and apoptosis. During the acute regenerative phase (first 24 hr) clusterin expression was markedly increased, decreasing to untraceable levels by five days of regeneration. Clusterin mRNA was localized in dilated or collapsed atrophic tubules that had lost identifying surface structures of normal tubular epithelium (termed dedifferentiated). Clusterin was also localized in the periphery of some blood vessel walls. Cell proliferation peaked at three to five days of regeneration, and was also localized in dedifferentiated tubules. Despite the regenerative stimulus, an unexpected result was a transient but marked increase in apoptotic cell death in atrophic tubules in the first 24 hours of regeneration. Our results provide evidence of a temporal association between increased clusterin expression and apoptosis, but in situ localization showed clusterin mRNA over apparently viable, as well as apoptotic, cells in the epithelium of tubules showing clusterin expression. Clusterin mRNA was rarely identified over epithelial cells in foci of non-atrophic (non-dedifferentiated) nephrons that responded to the regenerative stimulus by cellular hypertrophy. The dramatic response after initiation of regeneration, especially the initiation of apoptosis in the tubular epithelium, may have applications for the study of genetic changes leading to renal oncogenesis.

Animals↗
PubMed1994

Apoptosis in brain and gut tissue of mice fed a seed preparation of the cycad Lepidozamia peroffskyana.

Apoptosis (programmed cell death) was identified in histological sections of brain and gut tissue of adult mice fed seed preparations from the cycad Lepidozamia peroffskyana. This form of cell death was also found at high levels in brain tissue from neonatal mice born from a cycad-fed mother. The discovery was made during re-appraisal of archival tissue from a study of toxic properties of L. peroffskyana. Ingestion of appropriately prepared food or medicine derived from another cycad, Cycas circinalis, is thought to be associated with several motor neurone and other neurodegenerative disorders of some Pacific island inhabitants. Apoptosis is cell death under gene control. From the present study, presence of apoptosis in brain tissue after cycad toxicity may provide a link between cycad ingestion and development of neurodegenerative disorders and may provide a novel explanation for localization of some neurodegenerative disorders, as some inhabitants may have a genetic susceptibility to apoptosis induced by cycad toxicity.

Amyotrophic Lateral Sclerosis↗
PubMed1994

Expression and localization of sulphated glycoprotein-2 mRNA in the rat incisor tooth ameloblasts: relationships with apoptosis.

The expression of sulphated glycoprotein-2 (SGP-2) is associated with the onset of cellular atrophy and death in many rodent tissues. This gene has a multifunctional involvement that includes apoptosis, spermatogenesis, promotion of cell-cell interactions, modulation of complement systems and tissue regeneration and remodelling. Using decalcified mandibles, mRNA for SGP-2 in rat incisor tooth ameloblasts was examined by in situ hybridization using 35S riboprobes. The rat incisor is unique in that, at one time, all stages of the complex life cycle of the ameloblasts are represented along the length of the enamel forming aspect of the tooth. The pre-ameloblasts only secrete enamel matrix after mitosis. When the full thickness of the enamel has been formed, a remarkable transition in phenotype takes place in the ameloblast. This transition is accompanied by apoptosis or programmed cell death of approximately 25% of ameloblasts. An additional 25% of ameloblasts undergo apoptosis when maturation of enamel matrix takes place with removal of water and protein from the increasingly mineralized matrix. In the present study, expression of SGP-2 was localized most often in the post-secretory transition and maturation ameloblasts. In contrast, the presecretory and secretory ameloblasts did not demonstrate specific hybridization signals. Consistently, neither the odontoblasts nor the pulp demonstrated hybridization signals. Hence our results support other published results which show that increased expression of SGP-2 is associated with apoptosis. The exact function of the SGP-2 gene and its products is not fully defined.(ABSTRACT TRUNCATED AT 250 WORDS)

Ameloblasts↗
PubMed1993

Epstein-Barr virus infection is associated with increased apoptosis in untreated and phorbol ester-treated human Burkitt's lymphoma (AW-Ramos) cells.

Several papers have recorded that Epstein-Barr virus (EBV) infection can protect B-cells from apoptosis. In the present paper, we record an increase in apoptosis in an EBV-infected human Burkitt's lymphoma cell line (AW-Ramos), compared with its virus free counterpart (Ramos). The viral-infected cells died more rapidly by apoptosis during normal growth in culture: at 72h, Ramos cells had 8% apoptosis and AW-Ramos had 27% apoptosis. The EBV-infected cells were particularly sensitive to treatment with phorbol-12, 13-dibutyrate (PDBu, 0.05 microgram/ml): at 72h, Ramos cells had 12% apoptosis and AW-Ramos had 42% apoptosis. DNA gel electrophoresis supported the morphological findings. Our results serve as a caveat against generalizations that may be made about prevention of apoptosis by EBV infection.

Apoptosis↗
PubMed1992

Effects of hypoxia on morphological and biochemical characteristics of renal epithelial cell and tubule cultures.

Apoptotic cell death plays an important role in the pathogenesis of renal atrophy in diseases of the kidney involving chronic mild ischemia. The present study constitutes an in vitro model of these diseases and assesses the modes of cell death involved after hypoxic treatment of renal epithelium. Cultures of MDCK cells or primary cultures of rat renal parenchymal tubules were treated in either a physiological or a hypoxic atmosphere. Cultures were collected before treatment and at 24 h and 48 h, for morphological and biochemical studies. Both apoptosis and necrosis were observed at significantly increased levels by 48 h of hypoxia in the MDCK cell cultures. DNA gel electrophoresis patterns supported these findings. Experiments using tubule cultures demonstrated that, during the 48 h of study, tubular epithelial cells in the center of the control tubule structures died by apoptosis, possibly as a result of mild oxygen and/or nutrient depletion. With added hypoxic treatment, however, the entire tubule structure became necrotic. Results are similar to those found during in vivo studies, thus providing in vitro models that may be developed further to define factors in the pathogenesis of some renal diseases.

Animals↗
PubMed1991

The role of apoptosis in the response of cells and tumours to mild hyperthermia.

There is now abundant evidence that apoptosis, the cell death mechanism responsible for physiological deletion of cells, can be triggered by mild hyperthermia. However, the overall importance of this mode of death in heated tumours has not yet been established. In this light and electron microscopic study, apoptosis induced by 43 degrees C or 44 degrees C water bath heating for 30 min in a range of murine and human tumours growing in vitro and in four murine tumours growing as solid nodules in vivo, was identified on the basis of its characteristic morphology, and the amount present quantified. Apoptosis was found to play a variable role in the response of tumours to heating, with the lowest levels produced in human melanoma lines (less than 1%) and the highest levels in some Burkitt's lymphoma lines (up to 97%). In these latter tumours the induction of apoptosis is clearly a major component of the hyperthermic response.

Animals↗
PubMed1991

The role of apoptosis in the development of renal cortical tubular atrophy associated with healed experimental renal papillary necrosis.

An animal model of chronic analgesic nephropathy, in which renal papillary necrosis was induced by the administration of a single injection of bromoethylamine 2-hydrobromide in male Sprague-Dawley rats, was used to investigate the pathogenesis of the atrophy of tubules that leads to cortical atrophy or 'scarring' in analgesic nephropathy. One of the major objectives was documentation of the participation of apoptosis, a distinctive mode of cell death, in the process of cortical tubular atrophy. Control and treated groups of animals were studied at 2 wks, and at subsequent monthly intervals up to 4 mths. At each time, light microscopy and ultrastructure were used to relate changes in cellular pathology to alterations in renal mass. Apoptosis was quantitated in paraffin sections, and autoradiographic identification of cells showing tritiated thymidine uptake was used as an indication of cell proliferation. In animals with total renal papillary necrosis (RPN), focal or diffuse cortical atrophy developed, the extent of which appeared to be proportional to the extent of the RPN. Renal mass was reduced only in those kidneys that developed extensive, diffuse lesions. Compensatory renal growth occurred in the areas of healthy tissue adjacent to the foci of atrophy, with both cellular hyperplasia and hypertrophy playing roles in its development. One of the prominent cellular events was the appearance of apoptotic cells and bodies, with invading intraepithelial macrophages involved in their phagocytosis and degradation. We propose that this form of cell death plays an important role in the pathogenesis of cortical atrophy. Current descriptions of the cortical lesions that occur in analgesic nephropathy refer to the changes as 'scars'. Although the focal lesions have a macroscopic appearance that resembles scars, the results of the present study indicate that usage of this terminology may be misleading, since scarring is often described after severe tissue injury or necrosis, which was not identified in the present study.

Animals↗
PubMed1990

Cell death induced in a murine mastocytoma by 42-47 degrees C heating in vitro: evidence that the form of death changes from apoptosis to necrosis above a critical heat load.

The pathogenesis of heat-induced cell death is controversial. Categorizing the death occurring after various heat loads as either apoptosis or necrosis might help to elucidate this problem, since it has been shown that these two processes differ in their mode of initiation as well as in their morphological and biochemical features. Log-phase cultures of mastocytoma P-815 x 2.1 were heated at temperatures ranging from 42 to 47 degrees C for 30 min. After 42 degrees C heating a slight increase in apoptosis was observed morphologically. However, after heating at 43, 43.5 and 44 degrees C, there was marked enhancement of apoptosis, and electrophoresis of DNA showed characteristic internucleosomal cleavage. With heating at 45 degrees C both apoptosis and necrosis were enhanced, whereas at 46 and 47 degrees C only necrosis was produced. DNA extracted from the 46 and 47 degrees C cultures showed virtually no degradation, which contrasts with the random DNA breakdown observed in necrosis produced by other types of injury; lysosomal enzymes released during heat-induced necrosis may be inactivated at the higher temperatures. It is suggested that apoptosis following heating may be triggered either by a limited increase in cytosolic calcium levels resulting from mild membrane changes or by DNA damage. Necrosis, on the other hand, is likely to be a consequence of severe membrane disruption.

Animals↗