Leptin and its receptor: can they help to differentiate chromophobe renal cell carcinoma from renal oncocytoma?

One of the challenges in differentiating chromophobe renal cell carcinoma (chRCC) from benign renal oncocytoma (RO) is overlapping morphology between the two subtypes. The aim of this study was to investigate the usefulness of expression of leptin (Ob) and its receptor (ObR) in discriminating chRCC from RO. Sections from paraffin-embedded, formalin-fixed tumour nephrectomy specimens of 45 patients, made up of 30 chRCC (15 eosinophilic variant and 15 non-eosinophilic variant) and 15 RO, were used in this study. Samples (30) of clear cell RCC (ccRCC), the most common histological subtype, were used to verify staining patterns found by others in our cohort of Australasian patients. Matched morphologically normal non-cancer kidney tissues were included for each specimen. Sections were batch-immunostained using antibodies against Ob and ObR. Stained sections were digitally scanned using Aperio ImageScope, and the expression pattern of Ob and ObR was studied. In this cohort, male to female ratio was 2:1; median age was 64 (45-88 years); and median tumour size was 3.8 cm (range 1.2-18 cm). There were 47 (62.7%) T1, seven T2, 20 T3 and one T4 stage RCC. Two patients with ccRCC presented with metastases. Nuclear expression of Ob was significantly higher in RO compared with chRCC. The increased nuclear expression of Ob in RO compared with chRCC may be a useful aid in the difficult histological differentiation of RO from chRCC, especially eosinophilic variants of chRCC.

Therapeutic potential of abalone and status of bioactive molecules: A comprehensive review.

Marine organisms are increasingly being investigated as sources of bioactive molecules with therapeutic applications as nutraceuticals and pharmaceuticals. In particular, nutraceuticals are gaining popularity worldwide owing to their therapeutic potential and incorporation in functional foods and dietary supplements. Abalone, a marine gastropod, contains a variety of bioactive compounds with anti-oxidant, anti-thrombotic, anti-inflammatory, anti-microbial, and anti-cancer activities. For thousands of years different cultures have used abalone as a traditional functional food believing consumption provides health benefits. Abalone meat is one of the most precious commodities in Asian markets where it is considered a culinary delicacy. Recent research has revealed that abalone is composed of many vital moieties like polysaccharides, proteins, and fatty acids that provide health benefits beyond basic nutrition. A review of past and present research is presented with relevance to the therapeutic potential of bioactive molecules from abalone.

Granzyme M has a critical role in providing innate immune protection in ulcerative colitis.

Inflammatory bowel disease (IBD) is an immunoregulatory disorder, associated with a chronic and inappropriate mucosal immune response to commensal bacteria, underlying disease states such as ulcerative colitis (UC) and Crohn's disease (CD) in humans. Granzyme M (GrzM) is a serine protease expressed by cytotoxic lymphocytes, in particular natural killer (NK) cells. Granzymes are thought to be involved in triggering cell death in eukaryotic target cells; however, some evidence supports their role in inflammation. The role of GrzM in the innate immune response to mucosal inflammation has never been examined. Here, we discover that patients with UC, unlike patients with CD, display high levels of GrzM mRNA expression in the inflamed colon. By taking advantage of well-established models of experimental UC, we revealed that GrzM-deficient mice have greater levels of inflammatory indicators during dextran sulfate sodium (DSS)-induced IBD, including increased weight loss, greater colon length reduction and more severe intestinal histopathology. The absence of GrzM expression also had effects on gut permeability, tissue cytokine/chemokine dynamics, and neutrophil infiltration during disease. These findings demonstrate, for the first time, that GrzM has a critical role during early stages of inflammation in UC, and that in its absence colonic inflammation is enhanced.

Tumour necrosis factor receptor-associated factor-1 (TRAF-1) expression is increased in renal cell carcinoma patient serum but decreased in cancer tissue compared with normal: potential biomarker significance.

Renal cell carcinoma (RCC) generally has a poor prognosis because of late diagnosis and metastasis. We have previously described decreased tumour necrosis factor receptor-associated factor-1 (TRAF-1) in RCC compared with paired normal kidney in a patient cohort in Australia. In the present study, TRAF-1 expression in clear cell RCC (ccRCC) and normal kidney was again compared, but in a cohort from University Malaya Medical Centre. Serum TRAF-1 was also evaluated in RCC and normal samples.Immunohistochemistry with automated batch staining and Aperio ImageScope morphometry was used to compare TRAF-1 in 61 ccRCC with paired normal kidney tissue. Serum from 15 newly diagnosed and untreated ccRCC and 15 healthy people was tested for TRAF-1 using ELISA.In this cohort, TRAF-1 was highly expressed in proximal tubular epithelium of normal kidney, and significantly decreased in ccRCC tissue (p < 0.001). Conversely, TRAF-1 in serum from ccRCC patients was significantly increased over control serum (132 ±â€Š30 versus 54 ±â€Š14 pg/mL, respectively; p = 0.013).Decreased TRAF-1 in RCC tissue, reported previously, was confirmed. This, along with significantly increased serum TRAF-1 may indicate the protein is actively secreted during development and progression of ccRCC. Therefore, the increased serum TRAF-1 may be a useful non-invasive indicator of RCC development.

Renal cell carcinoma: resistance to therapy, role of apoptosis, and the prognostic and therapeutic target potential of TRAF proteins.

Renal cell carcinoma (RCC) is the commonest of the renal neoplasms. Although surgery and cryoablation are successful curative treatments for localized RCC, most patients are diagnosed with advanced or metastatic RCC, which has a poor prognosis. RCC are a heterogeneous set of cancers that have traditionally been classified and staged using cellular characteristics, size, local extension and distant metastases. Current staging systems provide good prognostic information, but it is very likely that the identification of new more accurate and predictive prognostic markers, not currently included in traditional staging systems, will improve the outcome for RCC patients. For this reason, increased knowledge of the underlying molecular characteristics of RCC development and progression is necessary. In most cancers, but especially RCC, deregulated control of apoptosis contributes to cancer growth by aberrantly extending cell viability and facilitating resistance to cancer therapies. Here we present the hypothesis that select members of the tumor necrosis factor (TNF) superfamily, the TNF receptor-associated factors (TRAFs), have a role in RCC apoptosis and may have prognostic significance for RCC. Candidate biomarkers for RCC are few, and the TRAFs may be important inclusions in panels of biomarkers for RCC. TRAFs may also be potential molecular targets for new therapies, either through their ability to promote apoptosis in the cancers themselves, or through their ability to modulate the immune defence against cancer progression. Some support data are presented here for our hypothesis. However, these novel concepts need further careful analysis to allow clinicians and oncologists any assistance for earlier detection of RCC and for characterizing patients with RCC for individualised targeted therapy.

Obesity and hypertension have differing oxidant handling molecular pathways in age-related chronic kidney disease.

Chronic kidney disease (CKD) in ageing is a burden on health systems worldwide. Rat models of age-related CKD linked with obesity and hypertension were used to investigate alterations in oxidant handling and energy metabolism to identify gene targets or markers for age-related CKD. Young adult (3 months) and old (21-24 months) spontaneously-hypertensive (SHR), normotensive Wistar-Kyoto (WKY) and Wistar rats (normotensive, obese in ageing) were compared for renal functional and physiological parameters, renal fibrosis and inflammation, oxidative stress (hemeoxygenase-1/HO-1), apoptosis and cell injury (including Bax:Bcl-2), phosphorylated and non-phosphorylated forms of oxidant and energy sensing proteins (p66Shc, AMPK), signal transduction proteins (ERK1/2, PKB), and transcription factors (NF-kappaB, FoxO1). All old rats were normoglycemic. Renal fibrosis, tubular epithelial apoptosis, interstitial macrophages and myofibroblasts (all p<0.05), p66Shc/phospho-p66 (p<0.05), Bax/Bcl-2 ratio (p<0.05) and NF-kappaB expression (p<0.01) were highest in old obese Wistars. Expression of phospho-FoxO/FoxO was elevated in old Wistars (p<0.001) and WKYs (p<0.01). SHRs had high levels in young and old rats. Expression of PKB, phospho-PKB, ERK1/2 and phospho-ERK1/2 were significantly elevated in all aged animals. These results suggest that obesity and hypertension have differing oxidant handling and signalling pathways that act in the pathogenesis of age-related CKD.

Delayed administration of darbepoetin or erythropoietin protects against ischemic acute renal injury and failure.

Administration of human recombinant erythropoietin (EPO) at time of acute ischemic renal injury (IRI) inhibits apoptosis, enhances tubular epithelial regeneration, and promotes renal functional recovery. The present study aimed to determine whether darbepoetin-alfa (DPO) exhibits comparable renoprotection to that afforded by EPO, whether pro or antiapoptotic Bcl-2 proteins are involved, and whether delayed administration of EPO or DPO 6 h following IRI ameliorates renal dysfunction. The model of IRI involved bilateral renal artery occlusion for 45 min in rats (N = 4 per group), followed by reperfusion for 1-7 days. Controls were sham-operated. Rats were treated at time of ischemia or sham operation (T0), or post-treated (6 h after the onset of reperfusion, T6) with EPO (5000 IU/kg), DPO (25 mug/kg), or appropriate vehicle by intraperitoneal injection. Renal function, structure, and immunohistochemistry for Bcl-2, Bcl-XL, and Bax were analyzed. DPO or EPO at T0 significantly abrogated renal dysfunction in IRI animals (serum creatinine for IRI 0.17 +/- 0.05 mmol/l vs DPO-IRI 0.08 +/- 0.03 mmol/l vs EPO-IRI 0.04 +/- 0.01 mmol/l, P = 0.01). Delayed administration of DPO or EPO (T6) also significantly abrogated subsequent renal dysfunction (serum creatinine for IRI 0.17 +/- 0.05 mmol/l vs DPO-IRI 0.06 +/- 0.01 mmol/l vs EPO-IRI 0.03 +/- 0.03 mmol/l, P = 0.01). There was also significantly decreased tissue injury (apoptosis, P < 0.05), decreased proapoptotic Bax, and increased regenerative capacity, especially in the outer stripe of the outer medulla, with DPO or EPO at T0 or T6. These results reaffirm the potential clinical application of DPO and EPO as novel renoprotective agents for patients at risk of ischemic acute renal failure or after having sustained an ischemic renal insult.

Evidence for a non-antioxidant, dose-dependent role of alpha -lipoic acid in caspase-3 and ERK2 activation in endothelial cells.

Endothelial cell apoptosis contributes to atherosclerosis and may be exacerbated by oxidative stress. Results from clinical trials using antioxidant supplementation are equivocal and could be enhanced by antioxidants with additional non-antioxidant properties such as alpha -lipoic acid and alpha -tocopherol. The aim of this study was to investigate the effects of these antioxidants on cytoprotective pathways and endothelial apoptosis. Endothelial cells were incubated with alpha -lipoic acid and alpha -tocopherol, alone or in combination, prior to incubation with H(2)O(2) or staurosporine. alpha -lipoic acid pre-treatment alone increased caspase-3 activity in a dose-dependent manner. Both H(2)O(2) and staurosporine increased DNA fragmentation and caspase-3 activity and pre-treatment of cells with alpha -lipoic acid and/or alpha -tocopherol failed to prevent stress-induced apoptosis. Neither antioxidant treatments nor apoptotic inducers alone altered expressions of Bcl-2, Bax, HSP70 or pERK1/2 or pJNK. alpha -lipoic decreased pERK2 in staurosporine-treated cells in a dose-dependent manner. These findings indicate that pre-incubation with alpha -lipoic acid and alpha -tocopherol, alone or in combination, does not protect against oxidative- or non-oxidative-induced apoptosis in endothelial cells. Moreover, we have demonstrated a non-antioxidant, dose-dependent role of alpha -lipoic acid in caspase-3 and ERK2 activation. These data provide an insight and indicate caution in the use of high doses of alpha -lipoic acid as an antioxidant.

Role of p53, apoptosis, and cell proliferation in early stage Epstein-Barr virus positive and negative gastric carcinomas.

AIMS: Mechanisms of Epstein-Barr virus (EBV) associated gastric tumour development are incompletely understood. The interrelations between EBV infection, apoptosis, cell proliferation, and the expression of the tumour suppressor gene p53 was investigated in 133 early stage gastric carcinomas. METHODS: Tumour tissue was compared with paired non-tumour tissue. EBV encoded small RNAs (EBERs) determined EBV status. The apoptotic index (AI) was determined by morphology and verified biochemically. p53 and Ki-67 expression (cell proliferation) was assessed using immunohistochemistry. RESULTS: EBV was detected in 14.3% of the cases. Cell proliferation did not differ significantly between EBV positive and negative cancers. However, within both these groups, the p53 positive and negative subsets differed significantly (EBV positive group: 76.8% and 55.3% were p53 positive or negative cancers, respectively; p<0.05; EBV negative group: 65.2% and 51.7% were p53 positive or negative, respectively; p<0.005). The numbers of p53 expressing EBV positive and negative cases were significantly different (57.9% and 82.5%, respectively; p<0.05). Compared with cell proliferation, apoptosis was significantly lower in EBV positive versus negative cancers (AI of 4.36 and 6.50, respectively; p<0.01). The p53 positive and negative subsets also differed significantly in AI (EBV positive group: AI of 5.13 and 3.30 for p53 positive and negative cancers, respectively; p<0.05: EBV negative group: AI of 6.84 and 4.90 for p53 positive and negative cancers, respectively; p<0.05). CONCLUSIONS: These factors probably combine to promote development and progression of early stage gastric carcinomas and, at the same time, ensure the survival of EBV itself.

Epstein-Barr virus-mediated protection against etoposide-induced apoptosis in BJA-B B cell lymphoma cells: role of Bcl-2 and caspase proteins.

Epstein-Barr virus (EBV)-infected B cell lymphomas are resistant to apoptosis during cancer development and treatment with therapies. The molecular controls that determine why EBV infection causes apoptosis resistance need further definition. EBV-positive and EBV-negative BJA-B B cell lymphoma cell lines were used to compare the expression of selected apoptosis-regulating Bcl-2 and caspase proteins in EBV-related apoptosis resistance, after 8 hr or 18-24 hr etoposide treatment (80 microM). Apoptosis was quantified using morphology and verified with Hoechst 33258 nuclear stain and electron microscopy. Fluorescence activated cell sorting (FACS) was used to analyse effects on cell cycle of the EBV infection as well as etoposide treatment. Anti-apoptotic Bcl-2 and Bcl-XL, pro-apoptotic Bax, caspase-3 and caspase-9 expression and activation were analysed using Western immunoblots and densitometry. EBV-positive cultures had significantly lower levels of apoptosis in untreated and etoposide-treated cultures in comparison with EBV-negative cultures (p < 0.05). FACS analysis indicated a strong G2/M block in both cell sublines after etoposide treatment. Endogenous Bcl-2 was minimal in the EBV-negative cells in comparison with strong expression in EBV-positive cells. These levels did not alter with etoposide treatment. Bcl-XL was expressed endogenously in both cell lines and had reduced expression in EBV-negative cells after etoposide treatment. Bax showed no etoposide-induced alterations in expression. Pro-caspase-9 and -3 were seen in both EBV-positive and -negative cells. Etoposide induced cleavage of caspase-9 in both cell lines, with the EBV-positive cells having proportionally less cleavage product, in agreement with their lower levels of apoptosis. Caspase-3 cleavage occurred in the EBV-negative etoposide-treated cells but not in the EBV-positive cells. The results indicate that apoptosis resistance in EBV-infected B cell lymphomas is promoted by an inactive caspase-3 pathway and elevated expression of Bcl-2 that is not altered by etoposide drug treatment.

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.

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.

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.

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.

In the absence of Epstein-Barr virus infection, phorbol ester modulates apoptosis in cycloheximide-treated Burkitt's lymphoma (BJA-B) cells.

We have shown previously that cycloheximide (CHX), a potent protein synthesis inhibitor, induces high levels of apoptosis in Epstein-Barr virus free (EBV(-)) Burkitt's lymphoma (BJA-B) cells, with comparably reduced levels of apoptosis in the EBV positive (EBV(+)) cells. Modulation of CHX-induced apoptosis in EBV(-) and (+) B cells is reported here using concurrent treatment with phorbol ester (phorbol 12, 13-dibutyrate, PdBu). Cells were collected at 0, 3, 6, 12, 24 and 48 hours after treatment with (i) 1 microgram/ml CHX, (ii) 0.1 microgram/ml PdBu (1 hour pretreatment before 0 h), or (iii) CHX + PdBu (CHX added at 0 h, 1 hour after PdBu). Control cultures were untreated. Apoptotic, necrotic or viable cells were quantified using histological, ultrastructural and biochemical parameters. Protein synthesis was assessed using 35S-methionine incorporation. Intracellular calcium concentrations were measured using flow cytometry. PdBu alone had little effect on cell death: High levels of CHX-induced apoptosis in EBV(-) cells were significantly reduced by concurrent addition of PdBu (P < 0.005). In contrast, low levels of CHX-induced apoptosis in EBV(+) cells were not significantly altered by PdBu treatment. In EBV(-) cells, a negative relationship was observed between levels of apoptosis and calcium concentrations, whereas in EBV(+) cells, there was negligible correlation between these parameters. Thus high levels of CHX-induced apoptosis in EBV(-) cells occur via a PKC-dependent pathway, whereas CHX treatment of EBV(+) cells induces comparatively low levels of apoptosis that occur via a PKC-independent mechanism. The results application in the therapeutic intervention for cancers developing in association with EBV infection.

Evidence that "myofibroblast-like" cells are the cellular source of capsular collagen in hepatocellular carcinoma.

BACKGROUND/AIMS: The prognosis for patients with hepatocellular carcinoma is poor although tumour encapsulation has been associated with improved survival and disease-free rates. While the source of the tumour capsule is unclear, the major role that activated hepatic stellate cells play in the deposition of liver matrix in normal and diseased states suggests the possible involvement of these cells in tumour encapsulation. METHODS: Twenty-four liver tumours (seven encapsulated HCC, seven non-encapsulated HCC, 10 colorectal metastases) were studied. Activated hepatic stellate cells were identified by immunohistochemistry for alpha-smooth muscle actin (alpha-SMA) and in situ hybridization for pro-collagen alpha1 (I) mRNA. Collagen deposition was localized using Masson's trichrome stain. RESULTS: Pro-collagen alpha1 (I) mRNA co-localized to alpha-SMA positive hepatic stellate cells within the region of increased collagen deposition in (i) the tumour capsule of encapsulated HCC, and (ii) the tumour junction of non-encapsulated HCC and colorectal metastasis. In addition, there was marked peritumour expression of alpha-SMA and procollagen alpha1 (I) mRNA, which diminished with distance away from the tumour in all tumour groups. The degree of expression was greatest with encapsulated HCC, less with non-encapsulated HCC and least with colorectal metastasis. This contrasted with the absence of alpha-SMA expression in normal liver from the same patients. Within the tumours, colorectal metastases differed from HCC by demonstrating marked alpha-SMA expression and collagen deposition in the septa. CONCLUSIONS: Our findings demonstrate that activated hepatic stellate cells (i) are responsible for increased peritumour collagen production in non-encapsulated HCC and colorectal metastasis, and (ii) may be implicated in tumour capsule formation in HCC and metastasis stroma development. Thus, stellate cells may influence the local hepatic invasion by these tumours.

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.

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.

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.

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.