Esther Rabizadeh

Accumulation and drainage of hemin in the red cell membrane

The subject of hemin intercalation in red cell membranes and the correlation of the accumulated hemin level with the membrane pathology was studied. Methods which made use of dioxan and octan-2-ol mixtures to quantitate small amounts of hemin in membranes were developed. Applying these methods, hemin levels were measured in the cytoskeleton and the remaining lipid core of various red cell membranes. The amount of hemin, in both membrane fractions, was higher in pathological cells of sickle cell anemia and beta-thalassemia as compared to normal circulating cells. Correlation exists between the amount of the membrane-accumulated hemin and the severity of the disease. The level of hemin in the membrane was found to be age dependent, old cells in circulation accumulating more hemin than young cells. The level of hemin in all cells tested was much lower than the amount found previously to cause immediate hemolysis when applied externally (Kirschner-Zilber, I., Rabizadeh, E. and Shaklai, N. (1982) Biochim. Biophys. Acta 690, 20-30). This was explained by the differences between the process leading to immediate lysis and membrane changes recognized as pathological by the in-vivo sequestration mechanism. In search of a physiological mechanism which may drain the cell membrane from the hazardeous hemin, albumin, the main serum protein, was found capable of serving as an efficient agent for extracting hemin trapped in red cell membranes. It is suggested that under normal conditions albumin extracts enough hemin to leave the erythrocyte with unharmful hemin amounts, however, under pathological conditions greater amounts accumulate leading to a shorter cell life span.

The interaction of hemin and bilirubin with the human red cell membrane.

The incubation of 0.5% suspension of fresh normal erythrocytes with hemin or bilirubin resulted in substantial hemolysis. The amount of hemolysis achieved depended on the concentration of the lytic agents. In each concentration maximum hemolysis was reached within half an hour. The hemolytic effect was somewhat dependent on temperature. Comparison with the hemolytic effect of hemin on mice (Chau, A.C. and Fitch, C.D. (1980) J. Clin. Invest. 66, 856-858) showed that although both cells undergo hemolysis by hemin, the behaviour of each red cell type is different. Centrifugation and fluorescence quenching of membrane embedded probe revealed that both hemin and bilirubin bind to the red cell membrane, hemin having higher affinity. The reaction was found to be hydrophobic and therefore independent of ionic strength. The high affinity of the membrane for hemin was shown by its ability to compete successfully with globin for hemin. Electron microscopy of the red cells which underwent hemolysis indicated cell damage and some membrane destruction. Red cell ghosts were totally disrupted when saturated with hemin. These results suggest an explanation for hemolytic events occurring in cases such as elevation of serum bilirubin or abnormalities leading to hemin release by hemoglobin.

All three receptors for vascular endothelial growth factor (VEGF) are expressed on B-chronic lymphocytic leukemia (CLL) cells

B-chronic lymphocytic leukemia (B-CLL) cells have a long survival owing to an alteration in the normal pathways of apoptosis. CLL cells have been found to produce and secrete vascular endothelial growth factor (VEGF). In addition to its major role in angiogenesis, VEGF affects cell survival by interfering with apoptosis. The aim of the present study was to investigate the expression of the VEGF receptors VEGFR-1, VEGFR-2, and VEGFR-3 on B-CLL cells, singly and combined. B-CLL cells were isolated from peripheral blood drawn from patients with CLL. Total VEGF receptor, examined in 13 samples by flow cytometry was present in all cases with mean CD19+/VEGF+ expression of 76% (range 52-92%). Specific receptor expression, examined in 27 samples by immunocytochemical methods, was positive for VEGFR-1 in all 27 patients and for VEGFR-2 and VEGFR-3 in 26 (96%). These findings suggest that the VEGF transduction pathway may be very active in CLL cells, and both its paracrine and autocrine pathways may contribute to their enhanced survival.

Bcl-2 expression correlates positively with serum basic fibroblast growth factor (bFGF) and negatively with cellular vascular endothelial growth factor (VEGF) in patients with chronic lymphocytic leukaemia

A large proportion of B‐chronic lymphocytic leukaemia (B‐CLL) cells express the anti‐apoptotic protein Bcl‐2. Basic fibroblast growth factor (bFGF) has been shown to upregulate the expression of Bcl‐2 in B‐CLL cell lines. Vascular endothelial growth factor (VEGF) has been shown to enhance the survival of endothelial cells by upregulating the expression of Bcl‐2. In the present study, we measured serum and cellular levels of bFGF and VEGF in 85 patients with CLL using a commercial quantitative sandwich enzyme immunoassay technique. Levels of Bcl‐2 were also assayed concomitantly using Western blot analysis. The mean serum level of bFGF was 53·4 pg/ml (range 0–589) and that of VEGF 459·2 pg/ml (range 33–1793). The mean cellular level of bFGF was 158·3 pg/2 × 105 cells (range 0·8–841) and VEGF, 42·4 pg/2 × 105 cells (range 0–244). A high correlation was found between serum and cellular bFGF levels (P < 0·001), but not between the corresponding VEGF levels. Twenty‐nine of 69 patients (42%) evaluated for Bcl‐2 level, expressed it. The Bcl‐2 level was positively correlated with the serum bFGF level (P = 0·007). However, surprisingly there was a negative correlation between Bcl‐2 expression and intracellular VEGF level (P = 0·003). A positive correlation was also found between serum bFGF and disease follow‐up time and log white blood cell count. These findings indicate that in CLL there is a correlation between angiogenesis‐related factors and apoptosis‐related protein expression, and elevated bFGF levels may account for the elevated Bcl‐2 levels.

Microvessel density in chemosensitive and chemoresistant diffuse large B-cell lymphomas

Preliminary reports involving a number of different kinds of tumors have indicated that microvessel quantification may be useful in predicting disease outcome. The aim of this study was to examine the relationship between microvessel density (MVD) as a parameter of tumor angiogenesis and the response to chemotherapy in diffuse large B-cell (DLBC) lymphomas.A total of 36 DLBC lymphoma patients were evaluated, 23 of them with a chemosensitive; responsive disease (median survival 8 y) and 13 with a chemoresistant, refractory disease (median survival 8 months). Microvessel quantification was performed by immunohistochemical staining, using monoclonal antibodies against factor VIII related antigen (F8RA) and against platelet/endothelial cell adhesion molecule-CD31.We found that F8RA stained a significantly higher number of blood vessels (about 2.5 times more) than CD-31; 7 samples were not stained with CD-31 but were positive for F8RA. There was no significant difference between the density of microvessel staining of the two groups. In the chemosensitive DLBC lymphomas positive for F8RA, the mean number of microvessels stained was 54.5±36.1 per microscopic field (200×) examined (range 6–149) whereas in the chemoresistant group the corresponding mean number was 43.1±25.5 (range 11–94).F8RA appears to be more sensitive for staining DLBC lymphomas microvessels than CD-31. Our data demonstrate that there is no correlation between tumor MVD and response to chemotherapy in patients with DLBC lymphomas.

Ovarian minimal residual disease in chronic myeloid leukaemia

The options for fertility preservation include cryopreservation of ovarian tissue. Although transplantation of cryopreserved-thawed ovarian tissue in cancer survivors has resulted in live births, there is evidence of malignancy involvement in ovarian tissue, especially in leukaemia. The objectives of this study were to investigate the involvement of chronic myeloid leukaemia (CML) in ovaries by both pathological/immunohistochemical methods and PCR for the identification of the Philadelphia chromosome (BCR-ABL transcripts). The patient was a survivor of paediatric CML whose ovaries were cryopreserved. The patient became infertile and requested ovarian reimplantation in adulthood. Pathological examinations of ovarian tissue with immunohistochemical stainings, quantitative PCR and two-step nested PCR were applied to identify BCR-ABL transcripts. Despite the lack of positive pathological/immunohistochemical evidence, PCR and two-step nested PCR revealed that the ovary was contaminated by malignant minimal residual CML. Survivors of childhood CML may harbour minimal residual disease in the ovaries. This finding stresses the danger of reseeding cancer by ovarian grafting, especially in patients with leukaemia. If ovarian grafting is considered, reimplantation should be preceded by examination of ovarian samples both pathologically and by molecular techniques. On the basis of molecular findings, ovarian autografting was not recommended in this case report.

Effect of the cytostatic butyric acid pro-drug, pivaloyloxymethyl butyrate, on the tumorigenicity of cancer cells

Previously we have shown that pivaloyloxymethyl butyrate (AN-9), a pro-drug of butyric acid (BA), is a differentiation-inducing agent in a variety of cells. In this report, we demonstrate that AN-9 is a cytostatic but not cytotoxic agent in a myelomonocytic cell line (WEHI); thus, the cells were growth-arrested and differentiated. These late changes in the cells were preceded by changes in the expression of the early regulatory genes,c-myc andc-jun. Although initiation of all these events had already occurred after 1 h exposure to AN-9, the tumorigenicity of these cells tested in Balb/c mice was not affected. A marked reduction in the tumorigenicity of AN-9-treated cells was observed after 4 h of exposure. Exposure of the highly metastatic subclone of Lewis lung carcinoma (3LLD122) to AN-9 resulted in a very pronounced effect on the tumorigenicity of these cells tested in C57BL mice. Unlike WEHI cells, the tumorigenicity of 3LLD122 was almost completely diminished after 1 h of exposure. In both cell types a 10-fold higher concentration of BA did not affect the tumorigenicity of the cells as did AN-9.

Effect of imatinib on the signal transduction cascade regulating telomerase activity in K562 (BCR-ABL-positive) cells sensitive and resistant to imatinib.

OBJECTIVE Imatinib mesylate (IM) is a tyrosine kinase inhibitor selective for BCR-ABL and indicated for the treatment of chronic myeloid leukemia. It has recently been demonstrated that IM also targets other cellular components. Considering the significant role of telomerase in malignant transformation, we studied the effect of IM on telomerase activity (TA) and regulation in BCR-ABL-positive and -negative cells, sensitive and resistant to IM. MATERIALS AND METHODS Through combining telomeric repeat amplification protocol for detecting TA, reverse transcription polymerase chain reaction and Western blots for detecting RNA and protein levels of telomerase regulating proteins and fluorescence-activated cell sorting analysis, we showed that IM targets telomerase and the signal transduction cascade upstream of it. RESULTS IM significantly inhibited TA in BCR-ABL-positive and -negative cells and in chronic myeloid leukemia patients. TA inhibition was also observed in BCR-ABL positive cells resistant to IM at drug concentrations that did not lead to a reduction in BCR-ABL expression. In addition, a reduction in phosphorylated AKT and phosphorylated PDK-1 was also detected following IM incubation. CONCLUSIONS We demonstrate an inhibitory effect of IM on TA and on the AKT/PDK pathway. Because this effect was observed in cell expressing the BCR-ABL protein as well as cells not expressing it, and in cells sensitive as well as resistant to IM, it is reasonable to assume that the inhibitory effect of IM on TA is not mediated through known IM targets. The results of this study show that cells resistant to IM with regard to its effect on BCR-ABL could still be sensitive to IM treatment regarding other cellular components.

Doxorubicin and a butyric acid derivative effectively reduce levels of BCL-2 protein in the cells of chronic lymphocytic leukemia patient

Abstract: B‐chronic lymphocytic leukemia (B‐CLL) is a disease caused primarily by defects in the apoptosis mechanism. AN‐9, a butyric acid (BA) derivative, is a potent differentiating and an anti‐cancer drug that induces apoptosis in HL‐60 cells. Herein we show the affect of AN‐9, alone and in combination with doxorubicin, on cell cultures from B‐CLL patients. Cells from 17 patients were cultured and tested for viability, apoptosis, bcl‐2 and bax protein expression. Exposure of B‐CLL cell cultures to AN‐9 was accompanied by apoptosis and a marked viability loss (up to 46%, p=0.0017). AN‐9 reduced up to 51% (p=0.0017) the levels of bcl‐2 in 57% of the cultures that express bcl‐2. The combination of low concentrations of AN‐9 and doxorubicin more than additively enhanced apoptosis and reduced bcl‐2 levels in B‐CLL cultures which were resistant to AN‐9. AN‐9 enhanced bax expression up to 58%(p=0.008) in cultures from 53% of the patients, but had no effect on bax levels when combined with doxorubicin.

Enteral n -3 fatty acids and micronutrients enhance percentage of positive neutrophil and lymphocyte adhesion molecules: a potential mediator of pressure ulcer healing in critically ill patients

n-3 Fatty acids are recognised as influencing both wound healing and immunity. We assessed the impact of a fish oil- and micronutrient-enriched formula (study formula) on the healing of pressure ulcers and on immune function in critically ill patients in an intensive care unit. A total of forty patients with pressure ulcers and receiving nutritional support were enrolled (intervention group, n 20, received study formula; and a control group, n 20, received an isoenergetic formula). Total and differential leucocyte count and percentage of adhesion molecule positive granulocyte and lymphocyte cells (CD11a, CD11b, CD18 and CD49b) were measured on days 0, 7 and 14. Percentage of positive lymphocytes for CD54, CD49b, CD49d and CD8 were also measured on days 0, 7 and 14. The state of pressure ulcers was assessed by using the pressure ulcer scale for healing tool score on days 7, 14 and 28 of treatment. No between-group differences in patient demographics, anthropometry or diagnostic class were observed. Patients who received the study formula showed significant increases in the percentage of positive CD18 and CD11a lymphocytes and of CD49b granulocytes as compared to controls (P < 0·05). While the severity of pressure ulcers was not significantly different between the two groups on admission, severity increased significantly over time for the control group (P < 0·05), but not for the study group. The present study suggests that a fish oil- and micronutrient-enriched formula may prevent worsening of pressure ulcers and that this effect may be mediated by an effect on adhesion molecule expression.