Prognostic value of hepatocyte proliferative activity after transjugular intrahepatic portosystemic shunt.

OBJECTIVES: Previous data indicated that the proliferating cell nuclear antigen-labeling index (PCNA-LI) reflects the liver functional reserve in human liver cirrhosis. The aim of the study was to evaluate the hepatocyte proliferative activity as a marker for the outcome of patients after transjugular intrahepatic portosystemic shunt (TIPS). METHODS: Twenty-eight consecutive patients were electively treated with TIPS for recurrent variceal bleeding (n = 14), refractory ascites (n = 12), or hydrothorax (n = 2). PCNA immunostaining was analyzed on methanol-fixed, paraffin-embedded liver biopsies. RESULTS: After TIPS, six patients died within the first 3 months, eight other patients died later, two were transplanted, and 12 were alive at the time of analysis. Early death occurred in patients with refractory ascites (5/12) and/or in Child C patients (3/6). Among the evaluated variables, there was a statistical trend for the PCNA-LI to be lower in patients who died early after TIPS than in those having long term survival (1.55% vs 2.65%, p = 0.07). After TIPS insertion, the probability of remaining alive during the first 6 months of follow-up was significantly higher in patients with a preprocedural PCNA-LI > 2.9%. CONCLUSIONS: The PCNA-LI measured on liver biopsy before the TIPS procedure might be a pre-TIPS marker to discriminate those patients for whom TIPS is likely to be beneficial.

Early- and late-stage Kaposi's sarcoma-derived cells but not activated endothelial cells can invade de-epidermized dermis.

Whether Kaposi's sarcoma is a true neoplasm or a reactive endothelial cell outgrowth triggered by inflammatory cytokines remains unclear. In this study, we investigated the differential invasive properties of activated endothelial cells and Kaposi's sarcoma cells in a model of de-epidermized dermis, supplying the cells with matrix barriers similar to those found in vivo. Cells derived from early "patch-stage" and from late "nodular-stage" Kaposi's sarcoma lesions exhibited similar invasive properties, which indicates that cells with an invasive potential are present in the early stages of tumor development. Slow accumulation of the cells into the extracellular matrix, together with a low proliferation index and with expression of anti-apoptotic proteins, suggest that the progression of Kaposi's sarcoma may be related to escape from cell death rather than to increased proliferation. The Kaposi's sarcoma-Y1 cell line, which is tumorigenic in nude mice, also exhibited invasive properties. By contrast to the Kaposi's sarcoma-derived spindle cells, however, which were scattered between the collagen bundles, the Kaposi's sarcoma-Y1 cell population had a higher proliferation index and displayed a multilayer arrangement. Inflammatory cytokines and Kaposi's sarcoma cell supernatant could activate and stimulate the growth of human dermal microvascular endothelial cell, but could not induce their invasion in this model, showing that activated endothelial cells do not fit all the requirements to traverse the various barriers found in the dermal extracellular matrix. These results confer to Kaposi's sarcoma cells a tumor phenotype and suggest that the in vivo dominant endothelial cell population represents a reactive hyperplasia rather than the true tumor process.

Iron induces Bcl-2 expression in human dermal microvascular endothelial cells.

Iron is suspected to be involved in the induction and/or progression of various human tumors. The present study was designed to investigate the effects of iron on endothelial cells, keeping in mind that the homeostasis of microvessels plays a critical role in neo-angiogenesis. Applying a model of human dermal microvascular endothelial cell terminal differentiation and death induced by serum deprivation, we found that iron salts (iron chloride and ferric nitrilotriacetate) provided a survival advantage to endothelial cells. Using immunohistochemistry and Western Blot analysis, we found that the extended cellular life span induced by iron was paralleled by an increase of Bcl-2 protein expression. Taken together, these observations suggest that iron may give a survival advantage to endothelial cells and represent a novel mechanism through which iron may contribute to tumorigenesis.

Iron chelators inhibit the growth and induce the apoptosis of Kaposi's sarcoma cells and of their putative endothelial precursors.

Iron is suspected to be involved in the induction and/or progression of various human tumors. More particularly, iron may be involved in the pathogenesis of Kaposi's sarcoma, a tumor of probable vascular origin. This study was designed to investigate the effect of iron deprivation on Kaposi's sarcoma. The effects of iron chelators and iron deprivation associated with serum withdrawal were investigated on Kaposi's sarcoma-derived spindle cells, on a transformed Kaposi's sarcoma cell line (Kaposi's sarcoma Y-1) and on endothelial cells, which are the probable progenitors of Kaposi's sarcoma cells. Desferrioxamine and deferiprone, two chemically unrelated iron chelators, induced a time- and concentration-dependent inhibition of endothelial and Kaposi's sarcoma cell growth. The inhibition of cell growth was associated with a decrease in Ki-67 and in both stable and total proliferating cell nuclear antigen expression. Inhibition of the progression through the G1-phase of the cell cycle was further evidenced by decreased expression of cyclin D1 and of p34 cyclin-dependent kinase 4. Terminal deoxynucleotidyl transferase-mediated desoxyuridinetriphosphate nick end labeling assay, flow cytometry with annexin-V-fluorescein and morphologic analysis indicated that iron chelation also induced a time- and concentration-dependent apoptosis. This apoptotic effect was prevented by the addition of exogenous iron. Induction of iron deprivation in the culture medium by serum withdrawal led to similar cell cycle effects, which, however, could only be partly reverted by the addition of exogenous iron. In conclusion, these results show that iron deprivation inhibits the growth and induces the apoptosis of Kaposi's sarcoma cells and of their putative endothelial precursors. This suggests that iron chelators may represent a potential therapeutic approach for the treatment of Kaposi's sarcoma.

Comparison of methods based on annexin-V binding, DNA content or TUNEL for evaluating cell death in HL-60 and adherent MCF-7 cells.

HL-60 and MCF-7 cells were treated with 0.15 microM camptothecin (CPT) or with the solvent dimethylsulfoxide (DMSO) for the controls, for 2, 3 and 4 h or for 24, 48 and 72 h, respectively. The apoptotic index (AI) was then evaluated in parallel by the following flow cytometric methods: (1) double staining of unfixed cells with fluoresceinated annexin V and propidium iodide (PI), this after detachment by trypsinization in the case of MCF-7 cultures; (2) prefixation in 70% ethanol, extraction of degraded, low molecular weight DNA with 0.2 M phosphatecitrate buffer and analysis of the DNA content stained with PI; (3) TUNEL, i.e. labelling of DNA strand breaks with biotin-dUTP, followed by staining with streptavidin-fluorescein and counterstaining with PI. In HL-60 cells, the three methods gave similar results for the AI (3-4% in the controls and at 2 h of CPT treatment, and 35-43% at 3 and 4 h after CPT). This indicates that CPT-induced membrane alteration and DNA fragmentation occurred concomitantly in those cells. For MCF-7 cells, CPT-induced apoptosis developed more slowly, the AI, whether based on annexin V or on DNA content, remained unchanged at 24 h, then was increasing to 8% at 48 h and to 25% at 72 h of treatment. In these cells, the TUNEL index did not increase prior to 72 h, and the increase was minor (up to 9% vs. 2-3% in the controls) at 72 h of the treatment. This indicates that in MCF-7 cells DNA strand breaks cannot be effectively labelled, which may be due to inaccessibility of 3'-OH ends in the breaks to exogenous terminal deoxynucleotidyl transferase. The mechanism of endonucleolytic DNA fragmentation thus may be different, depending on the cell type.

Hepatocyte proliferative activity in human liver cirrhosis.

BACKGROUND/AIMS: The objective of this study was to validate, with an independent prospective cohort of patients, our previous data indicating that the proliferating cell nuclear antigen-labeling index (PCNA-LI) reflects the liver functional reserve in human cirrhosis and might have prognostic significance for patient survival. We also examined how this proliferative index is related to the expression of transforming growth factor beta1 (TGFbeta1) as a possible correlate of hepatocyte proliferative activity. METHODS: The present group (n=70 patients) was similar in composition to our previous group regarding age, sex and severity of liver cirrhosis. PCNA and TGFbeta1 immunostaining were analyzed on methanol-fixed, paraffin-embedded liver biopsies. RESULTS: Our data show that PCNA-LI declined significantly with worsening Child class and was negatively correlated with the Pugh score. Twenty-five patients died and 10 underwent liver transplantation during the observation period. Liver function, hepatic venous pressure gradient and hepatocyte PCNA-LI were significantly different in survivors and non-survivors. At a mean follow-up of 356 days, the patients with a PCNA-LI higher than 4.4% (the previously determined best cut-off value) had a significantly higher probability of survival than those with a PCNA-LI < or = 4.4% (0.87 vs 0.48, p=0.0009). TGFbeta1 expression in liver parenchyma correlated negatively with PCNA-LI, suggesting that this cytokine could be involved in the impaired regeneration observed in worsened liver cirrhosis. CONCLUSIONS: This prospective study strengthens our previous observation that, in cirrhosis, hepatocyte proliferative activity, as evaluated by the PCNA-LI, provides information on liver functional reserve as well as on the patient's prognosis.

Interleukin-10 controls neutrophilic infiltration, hepatocyte proliferation, and liver fibrosis induced by carbon tetrachloride in mice.

The role of the anti-inflammatory cytokine interleukin-10 (IL-10) was investigated in the mouse model of liver injury induced by carbon tetrachloride (CCl4). To address the role of endogenous IL-10 production, acute hepatitis was induced by CCl4 in C57Bl/6 IL-10 gene knock out (KO) and wild-type (WT) mice. After CCl4 challenge, serum and liver levels of tumor necrosis factor-alpha (TNF-) and serum levels of transforming growth factor-beta 1 (TGF-beta1) increased and were significantly higher in IL-10 KO mice, whereas IL-6 serum levels were only slightly increased compared with WT mice. At histological examination, the livers disclosed a significantly more prominent neutrophilic infiltration in IL-10 KO mice 12 and 24 hours after CCl4 injection. In contrast, hepatocyte necrosis, evaluated by histological examination and serum alanine aminotransferase levels, was only marginally affected. The proliferative response of hepatocytes, assessed by the proliferating cell nuclear-antigen labeling index, was significantly increased in IL-10 KO mice, compared with WT mice 48 hours after CCl4 injection. Finally, repeated CCl4 injections led to more liver fibrosis in IL-10 KO mice after 7 weeks. In conclusion, endogenous IL-10 marginally affects the hepatocyte necrosis although it controls the acute inflammatory burst induced by CCl4. During liver repair, it limits the proliferative response of hepatocytes and the development of fibrosis.

Overexpression of Bcl-2 in Kaposi's sarcoma-derived cells.

The pathogenesis of Kaposi's sarcoma (KS), a tumor of probable vascular origin, remains an enigma. It is still unclear whether KS is a true malignancy or whether it represents a reactive polyclonal process. Using both an immunohistochemical and an immunoblot approach, we found that cells derived from KS lesions express significant levels of Bcl-2, a protein known to prolong cellular viability and to antagonize apoptosis. Bcl-2 expression was found in AIDS-related KS-derived cells, as well as in cells derived from iatrogenic and sporadic KS, indicating that Bcl-2 upregulation may be important in the pathogenesis of KS regardless of its epidemiologic form. By contrast, fibroblasts and dermal microvascular endothelial, cells which are the probable vascular progenitors of KS cells, expressed low levels of Bcl-2. The expression of Bcl-2 in KS-derived cells was associated with a long-term survival in serum-deprived conditions, a situation that has been shown to induce apoptosis in various cell types. Incubation of fibroblasts or of dermal microvascular endothelial cells with KS cell-free supernatants did not enhance Bcl-2 expression, suggesting that Bcl-2 expression is not mediated by an agent released by KS cells. Analogously, KS supernatants failed to promote the viability of fibroblasts and of dermal microvascular endothelial cells cultured in serum-free conditions. Our findings suggest that the spindle cells derived from KS have a survival advantage and may adequately represent the tumor cells of KS.

Relationship between hepatocyte proliferative activity and liver functional reserve in human cirrhosis.

Hepatocyte proliferative activity is elevated in cirrhotic patients who develop hepatocellular carcinoma (HCC) and decreased in alcohol-induced hepatitis patients with poor outcome. Hepatocyte proliferative activity has not been evaluated in an unselected population of cirrhotic patients regarding the severity of the disease. Forty-six cirrhotic patients (21 alcoholic, 20 viral, and 5 other) were prospectively analyzed by proliferating cell nuclear antigen (PCNA) immunostaining on methanol-fixed, paraffin-embedded liver biopsy specimens. In these conditions, the PCNA-labeling index (PCNA-LI) measures the number of cells in the S-phase and assesses tissue proliferation. The median value of the PCNA-LI for all samples was 4.3% (range, 0%-20.2%). It declined with worsening Child-Pugh score: 9.15% (range, 3.3%-20.2%), 5.3% (range, 1.2%-18%), and 2.4% (range, 0%-4.4%) in Child classes A, B, and C, respectively (P < .05). Using the best cutoff PCNA-LI value to divide cirrhosis into slowly and rapidly proliferating tissue subsets, the PCNA index was independently associated with serum albumin. The probability of survival in patients with a high PCNA-LI ( > 4.4%) was significantly higher than in those with a lower PCNA-LI (0.93 vs. 0.53, at a median follow-up of 153 days; P = .01). In all 6 patients undergoing placement of a transjugular intrahepatic portosystemic shunt (TIPS), the PCNA-LI decreased after the procedure. This early impairment of hepatocyte proliferative activity after TIPS placement might reflect the functional alterations induced by this treatment. In conclusion, hepatocyte proliferative activity assessed by PCNA-LI is increased in cirrhotic patients and decreases with worsening of the disease.

PCNA immunopositivity index as a substitute to 3H-thymidine pulse-labeling index (TLI) in methanol-fixed human lymphocytes.

PHA-stimulated human lymphocytes or myelogenous leukemia cells (strain K-562) were pulse labeled with 3H-thymidine and submitted to various fixation-permeabilization procedures. They were then immunostained with the 19A2, 19F4 or PC10 monoclonal antibody against the proliferating cell nuclear antigen (PCNA). The preparations were finally scored for the proportion of unlabeled, double-labeled and single PCNA or 3H-thymidine-labeled nuclei. Unstimulated lymphocytes were immunonegative in all the conditions tested, as also were stimulated lymphocytes checked with an isotype of the primary antibody. A specificity (Sp) and a sensitivity (Se) score was calculated to evaluate the recognition by PCNA staining of the S-phase cells, as defined by the 3H-labeling. The data show that in most instances the three antibodies recognized the 3H-labeled cells with high sensitivity, ie with few false negative, but with low specificity, ie with PCNA positivity extending to variable proportions of non-S-phase cells. By contrast, methanol fixation followed by a brief treatment with the detergent Triton X-100 and immunostaining with either 19F4 or PC10 (but not with 19A2) combined a high sensitivity and specificity scores of the recognition of the 3H-thymidine-labeled cells: PC10 gave a more intense and, hence, more readable reaction. PHA-stimulated lymphocytes that had been preserved at -20 degrees C as cytocentrifuged smears failed to show any immunopositivity for PCNA if not submitted to further fixation prior to the immunocytochemical assay. When methanol-Triton was used for this step, only PC10 gave positive immunoreaction, yet with a lower specificity score (Sp = 76%) than in cells submitted to this fixation-permeabilization procedure without prior cryopreservation (Sp = 91.7%). The PCNA index was measured in cryopreserved, methanol-fixed smears of lymphocytes from patients with various hematological diseases and was compared to the Ki-67 index established independently on a serial sample. A good correlation was found between the two indices (r = 0.79; P < 0.0001) with the PCNA index generally lower than or close to the Ki-67 index. This warrants a note of caution about the use of total (ie stable and labile) PCNA immunostaining to measure the growth fraction (GF), classically defined as the proportion of proliferating cells in a population. However, in the absence of an absolute reference marker for G0 cells, there is no reason to assume that the PCNA index would necessarily be a worse estimate of GF than the Ki-67 index.(ABSTRACT TRUNCATED AT 400 WORDS)

Identification of the cellular protein encoded by the human Wilms' tumor (WT1) gene.

A putative tumor-suppressor gene (wt1) located at chromosome 11p13 and involved in Wilms' tumor development has recently been identified as a zinc finger polypeptide-encoding gene. The purpose of this study was to characterize the protein encoded by the human wt1 gene. The region spanning the entire zinc finger domain was amplified by polymerase chain reaction (PCR) and subcloned in the pATH 3 expression vector. Polyclonal antibodies against the fused TrpE-WT protein were raised. These antibodies immunoprecipitated a 49- to 51-kDa protein from hematopoietic tumor cells labeled in vivo with [35S]methionine. Subcellular fractionation and immunohistochemistry followed by confocal microscopy indicated that the Wilms' tumor gene product (WT1) is mainly localized within the nucleus.

S phase duration measurement by combined PCNA/cyclin immunostaining and radioautography after a single pulse-labelling with 3H-thymidine.

A method for measuring S phase duration is described and evaluated that combines single pulse labelling with 3H-thymidine (TdR), detected by radioautography, and proliferating cell nuclear antigen (PCNA)/cyclin immunostaining to replace the second pulse labelling of the classical double-labelling method. Conditions were set up in which nuclei showing one or both types of label were readily distinguished, hence allowing to verify that cell fluxes in and out of S phase were equal. S phase durations thus measured in different tissues of the mouse were concordant with those obtained by the double 3H-TdR labelling or from labelled mitoses curves. Our method might be used with archived samples of methanol-fixed cells or tissues, singly labelled with 3H-TdR or with bromodeoxyuridine.

Cyclin/PCNA immunostaining as an alternative to tritiated thymidine pulse labelling for marking S phase cells in paraffin sections from animal and human tissues.

Paraffin sections from animal or human tissues fixed in different fixatives were submitted to immunostaining with the mouse monoclonal antibody 19A2, developed by Ogata et al. (1987a) against cyclin/PCNA. Detection of the bound antibody was performed by the indirect method with biotinylated sheep antibody and streptavidin-biotin-peroxidase complexes. No, or faint, nuclear staining was seen in material fixed in ethanol, Bouin, Bouin-Hollande, Carnoy or formaldehyde, whereas readily detectable immunocytochemical reaction was constantly observed over nuclei of methanol-fixed tissues. Hydrolysis with 2 N HCl prior to immunocytochemistry (as currently performed to render incorporated BrdU accessible to antibodies) somewhat improved the results with Bouin or Carnoy and markedly augmented the intensity of the peroxidase reactions in formaldehyde and in methanol-fixed tissues. The distribution of the positive nuclei in the two latter cases coincided with the proliferative compartment. On the other hand, double labelling with [3H]-thymidine and with the cyclin/PCNA antibody revealed that in methanol-fixed tissues the cyclin/PCNA labelling index did not differ by more than 6% from the [3H]-thymidine index. Besides the two labels overlapped in a proportion of labelled cells that was in reasonable agreement with expectation considering cells flow in and out of S phase since the time of [3H]-thymidine injection. This indicates that both labels recognize the same cells in this material. In contrast, in formaldehyde-fixed tissues, the cyclin/PCNA labelling index markedly exceeded the [3H]-thymidine labelling index. From this it is concluded that cyclin/PCNA immunostaining can be used: (1) In formaldehyde-fixed tissues (including existing material stored as paraffin blocks): for defining and mapping the proliferative (or germinative) compartment. (2) In methanol-fixed tissues as a substitute to the [3H]-thymidine autoradiographic labelling index. From this, a method is proposed (derived from classical 'double-labelling' technique) for measuring S phase duration in tissues fixed at a known interval time after a single labelling with [3H]-thymidine (or BrdU) and submitted to cyclin/PCNA immunocytochemical detection and to autoradiography (or to BrdU immunostaining).