Activation of Evi1 inhibits cell cycle progression and differentiation of hematopoietic progenitor cells.

The transcription factor Evi1 has an outstanding role in the formation and transformation of hematopoietic cells. Its activation by chromosomal rearrangement induces a myelodysplastic syndrome with progression to acute myeloid leukemia of poor prognosis. Similarly, retroviral insertion-mediated upregulation confers a competitive advantage to transplanted hematopoietic cells, triggering clonal dominance or even leukemia. To study the molecular and functional response of primary murine hematopoietic progenitor cells to the activation of Evi1, we established an inducible lentiviral expression system. EVI1 had a biphasic effect with initial growth inhibition and retarded myeloid differentiation linked to enhanced survival of myeloblasts in long-term cultures. Gene expression microarray analysis revealed that within 24 h EVI1 upregulated 'stemness' genes characteristic for long-term hematopoietic stem cells (Aldh1a1, Abca1, Cdkn1b, Cdkn1c, Epcam, among others) but downregulated genes involved in DNA replication (Cyclins and their kinases, among others) and DNA repair (including Brca1, Brca2, Rad51). Cell cycle analysis demonstrated EVI1's anti-proliferative effect to be strictly dose-dependent with accumulation of cells in G0/G1, but preservation of a small fraction of long-term proliferating cells. Although confined to cultured cells, our study contributes to new hypotheses addressing the mechanisms and molecular targets involved in preleukemic clonal dominance or leukemic transformation by Evi1.

Cell cycle-dependent AgNOR analysis in invasive breast cancer.

OBJECTIVE: To investigate to what extent analysis of silver-stained nucleolar organizer regions (AgNORs) is cell cycle dependent in breast cancer and to assess the prognostic value of an AgNOR analysis that takes into consideration the cell cycle status of tumor cells. STUDY DESIGN: In 97 cases of invasive breast carcinoma, morphometric AgNOR analysis was performed in tumor cells with immunohistochemical MIB-1 reactivity (NORcyc analysis) and in MIB-1-negative tumor cells (NORnon analysis). Additionally, conventional (NORconv) analysis without preceding MIB-1 staining was done. Findings were compared with the Nottingham prognostic index (NPI). RESULTS: In comparison to noncycling tumor cells, cycling ones exhibited significantly higher AgNOR numbers (mean values, 3.84 +/- 1.09 vs. 2.40 +/- 0.78 per nucleus), higher total AgNOR areas (5.95 +/- 3.17 vs. 5.62 +/- 3.05 micron 2, NS) and significantly lower mean AgNOR areas (2.08 +/- 1.14 vs. 2.93 +/- 1.69 micron 2). When related to NPI, correlation coefficients of NORnon analysis were higher than those of NORcyc analysis but lower than those of NORconv analysis. Among the different AgNOR parameters, total AgNOR area correlated best with NPI. CONCLUSION: Cell cycle status has a high impact on AgNOR analysis. However, the best prognostic information in breast cancer is derived from an AgNOR analysis that considers both cycling and noncycling tumor cells.

Identification of MK-944a: a second clinical candidate from the hydroxylaminepentanamide isostere series of HIV protease inhibitors.

Recent results from human clinical trials have established the critical role of HIV protease inhibitors in the treatment of acquired immune-deficiency syndrome (AIDS). However, the emergence of viral resistance, demanding treatment protocols, and adverse side effects have exposed the urgent need for a second generation of HIV protease inhibitors. The continued exploration of our hydroxylaminepentanamide (HAPA) transition-state isostere series of HIV protease inhibitors, which initially resulted in the identification of Crixivan (indinavir sulfate, MK-639, L-735,524), has now yielded MK-944a (L-756,423). This compound is potent, is selective, and competitively inhibits HIV-1 PR with a K(i) value of 0.049 nM. It stops the spread of the HIV(IIIb)-infected MT4 lymphoid cells at 25.0-50.0 nM, even in the presence of alpha(1) acid glycoprotein, human serum albumin, normal human serum, or fetal bovine serum. MK-944a has a longer half-life in several animal models (rats, dogs, and monkeys) than indinavir sulfate and is currently in advanced human clinical trials.

Apical macropinocytosis in polarized MDCK cells: regulation by N-ethylmaleimide-sensitive proteins.

In cells tested so far endocytosis seems to be dependent on N-ethylmaleimide (NEM)-sensitive proteins, and treatment with NEM results in a complete block of endocytosis. We here demonstrate that treatment of polarized MDCK I cells with NEM strongly increased endocytosis of ricin and horseradish peroxidase at the apical side, and electron microscopy revealed NEM-induced formation of large macropinosomes at the apical pole. The NEM-stimulated apical endocytosis seemed to involve phosphatidylinositol-3 kinase, protein kinase C and phospholipase D and it was dependent on ATP. Moreover, in contrast to endocytosis in nonpolarized cells ricin endocytosis at the basolateral side continued in the presence of NEM whereas endocytosis of transferrin was blocked. Furthermore, recycling of ricin endocytosed in the absence of NEM was not inhibited on either side upon addition of NEM demonstrating the existence of a NEM-resistant fusion machinery. The results suggest that the fusogenic property of both the apical and the basolateral plasma membrane of MDCK cells differs from that typically observed in cells unable to polarize.

Identification of filamin as a novel ligand for caveolin-1: evidence for the organization of caveolin-1-associated membrane domains by the actin cytoskeleton.

Reports on the ultrastructure of cells as well as biochemical data have, for several years, been indicating a connection between caveolae and the actin cytoskeleton. Here, using a yeast two-hybrid approach, we have identified the F-actin cross-linking protein filamin as a ligand for the caveolae-associated protein caveolin-1. Binding of caveolin-1 to filamin involved the N-terminal region of caveolin-1 and the C terminus of filamin close to the filamin-dimerization domain. In in vitro binding assays, recombinant caveolin-1 bound to both nonmuscle and muscle filamin, indicating that the interaction might not be cell type specific. With the use of confocal microscopy, colocalization of caveolin-1 and filamin was observed in elongated patches at the plasma membrane. Remarkably, when stress fiber formation was induced with Rho-stimulating Escherichia coli cytotoxic necrotizing factor 1, the caveolin-1-positive structures became coaligned with stress fibers, indicating that there was a physical link connecting them. Immunogold double-labeling electron microscopy confirmed that caveolin-1-labeled racemose caveolae clusters were positive for filamin. The actin network, therefore, seems to be directly involved in the spatial organization of caveolin-1-associated membrane domains.

Non-active site changes elicit broad-based cross-resistance of the HIV-1 protease to inhibitors.

Three high level, cross-resistant variants of the HIV-1 protease have been analyzed for their ability to bind four protease inhibitors approved by the Food and Drug Administration (saquinavir, ritonavir, indinavir, and nelfinavir) as AIDS therapeutics. The loss in binding energy (DeltaDeltaG(b)) going from the wild-type enzyme to mutant enzymes ranges from 2.5 to 4.4 kcal/mol, 40-65% of which is attributed to amino acid substitutions away from the active site of the protease and not in direct contact with the inhibitor. The data suggest that non-active site changes are collectively a major contributor toward engendering resistance against the protease inhibitor and cannot be ignored when considering cross-resistance issues of drugs against the HIV-1 protease.

A simple procedure for morphometric analysis of processes and growth cones of neurons in culture using parameters derived from the contour and convex hull of the object.

Morphometric estimation of neuronal processes is currently laborious and time-consuming, since the individual processes (axons and dendrites) have to be traced manually. In order to facilitate the measurement of cellular processes, we have tested a series of parameters derived from the contour and the convex hull of an object and estimated to which extent they reflect process length and number. The parameters included the area, perimeter and form factor of the object and convex hull, their ratios as well as object length, breadth, width, length/width and spreading index. Some new parameters derived from the contour and convex hull of the object, were also computed: process index (the number of areas contained within the convex hull outside the object contour), process domain (the total area contained within the convex hull outside the object contour), their ratio and the square root of the process domain (SR process domain). In total, 18 parameters were estimated. Populations of motoneurons, growth cones of cerebellar granule cells and N2a neuroblastoma cells were utilized due to their diversity in morphological features. The processes of each object were drawn by hand to establish the actual length and number. Total process length per object correlated strongly with object perimeter, process domain and SR process domain. The number of processes per object correlated well with perimeter ratio, process index and form factor, whereas object length, convex hull perimeter and spreading index correlated acceptably with the average process length. Using these parameters for the evaluation of neurite outgrowth in developing of hippocampal neurons in vitro, variables such as object perimeter, process domain and SR process domain were found to be very well suited for estimation of the total length of neurites. We conclude that based on the contour and convex hull of an object it is possible to calculate a series of parameters which may substitute direct measurements of process length.

NCAM-fibronectin-type-III-domain substrata with and without a six-amino-acid-long proline-rich insert increase the dendritic and axonal arborization of spinal motoneurons.

The neural cell adhesion molecule (NCAM) is a modulator of neurite outgrowth in vitro and in vivo. To see if single or tandem extracellular NCAM domains can influence neurite outgrowth, motoneurons from embryonic rat spinal cord were cultured on several NCAM fusion protein substrata. Motoneurons growing on either of two fusion proteins comprising the combined two fibronectin type III homology domains of NCAM with or without a six-amino-acid-long, proline-rich insert (F3I,II+ and F3I,II, respectively) usually developed three or more neurites per cell. Motoneurons grown on NCAM-immunoglobulin domain I (IgI), by contrast, developed many unipolar and bipolar cells, a situation also seen when motoneurons were cultured on control substrata. The neuritic trees of motoneurons grown on F3I,II and F3I,II+ appeared broader and rounder than motoneurons cultured on either control or IgI substrata, and the spreading indices of motoneurons grown on F3I,II and F3I,II+ were significantly lower than when the other substrata were used. Neither of the NCAM-F3 fusion proteins stimulated the outgrowth of single neurites. By contrast, IgI substratum was able to stimulate neurite outgrowth over control substrata. Both NCAM-F3 substrata induced branches in axons and dendrites, whereas IgI substratum did not affect neurite branching significantly. These data indicated that neurite outgrowth and neurite branching on the chosen substrata were not closely linked to each other. Furthermore, the branching characteristics of motoneuron neurites potentially depend on their differentiation states and, possibly, on the conformation of the two NCAM-F3 domains.

Functional characterization of NCAM fibronectin type III domains: demonstration of modulatory effects of the proline-rich sequence encoded by alternatively spliced exons a and AAG.

In order to characterize the functions of the two fibronectin type III (F3) homology domains of the neural cell adhesion molecule (NCAM), we investigated the effects of two variants, expressed as fusion proteins, of the NCAM-F3 domains on attachment and spreading of NCAM-expressing fibroblasts, cerebellar cell aggregation and fiber formation, and on growth cones. The two fusion proteins were different with regard to a short proline-rich insert of six amino acids between the two F3 domains. Immobilized NCAM-F3 fusion proteins were found to mediate attachment of both transmembrane and lipid-anchored NCAM expressing fibroblasts. Also NCAM-negative cells adhered to the NCAM-F3 substratum, although to a lesser extent, implying the possibility of a heterophilic ligand to NCAM-F3 domains on the surface of fibroblasts. Cellular spreading on NCAM-F3 substratum was selectively increased in fibroblasts expressing transmembrane NCAM, and only the NCAM-F3 fusion protein lacking the proline-rich insert was able to elicit this effect. Primary cultures of mouse cerebellum were strongly inhibited with regard to formation of cellular aggregates and fibers, when incubated in the presence of either of the two NCAM-F3 fusion proteins, the fusion protein with the proline-rich insert being the more effective one. Finally, the morphology of growth cones from rat cerebellar granule cells changed significantly when grown on NCAM-F3 substrata as revealed by computer-assisted image analysis. Thus, our data indicate that the NCAM-F3 domain are involved in cell-cell adhesion, and that insertion of the proline-rich sequence has a modulatory effect on NCAM-F3 domain functions.

Elucidation of basic mechanistic and kinetic properties of influenza endonuclease using chemically synthesized RNAs.

Influenza virus utilizes a unique mechanism for initiating the transcription of viral mRNA. The viral transcriptase ribonucleoprotein complex hydrolyzes host cell transcripts containing the cap 1 structure (m7GpppG(2'-OMe)-) to generate a capped primer for viral mRNA transcription. Basic aspects of this viral endonuclease reaction are elucidated in this study through the use of synthetic, radiolabeled RNA substrates and substrate analogs containing the cap 1 structure. Unlike most ribonucleases, this viral endonuclease is shown to catalyze the hydrolysis of the scissile phosphodiester, resulting in 5'-phosphate- and 3'-hydroxyl-containing fragments. Nevertheless, the 2'-OH adjacent to the released ribosyl 3'-OH is shown to be important for catalysis. In addition, while the endonuclease steady-state turnover rate is measured to be 2 h(-1), phosphodiester bond hydrolysis is not rate-limiting. The direct generation of a free 3'-OH and the subsequent slow release of this product are consistent with the viral need for efficient use of the capped primer in subsequent reactions of the influenza transcriptase complex.

Expression and purification of retroviral HIV-1 reverse transcriptase.

Modern molecular biology techniques have provided valuable tools which allow for the expression of large amounts of enzyme in E. coli. For potential therapeutic targets such as HIV-1 reverse transcriptase, it is desirable that the enzyme studied is pure and correlates to the active form of the enzyme found in vivo. This poses a particular challenge for those researchers studying HIV-RT since a significant degree of heterogeneity is introduced by nonspecific proteolytic cleavage of the p66 subunit by E. coli proteases. The advantage of the purification protocol presented here is that the association of monomers is facilitated by mixing an excess of p51 subunit, which is truncated at a site that is N-terminal to known bacterial cleavage sites, with p66 protein. This avoids enzymatic processing of the larger subunit since the formation of heterodimeric RT is rapid and the dimer is stable against proteolytic cleavage. Therefore, it is possible to isolate a pure homogeneous p66/p51 heterodimer. An enzyme prepared in this manner yields crystals that defract to a 3.2-A resolution. It has also been used to study both sensitivity of HIV-1 RT mutants to azidothymidine triphosphate and the kinetics of a potent nonnucleoside RT inhibitor (L-743,726). Finally, it is interesting to note the similarity of HIV-1 RT with reverse transcriptases from other lentiviruses (FIV and EIAV RT). Both of these enzymes consist of heterodimers of p66 and p51 subunits and share other biophysical characteristics. Purification of these reverse transcriptases can, in all likelihood, be optimized by using methods similar to those described in this chapter.

Purification and characterization of HIV-1 reverse transcriptase having a 1:1 ratio of p66 and p51 subunits.

Wild-type and several mutant forms of recombinant human immunodeficiency virus type-1 reverse transcriptase were overexpressed as either the p66 or the p51 subunit in a protease-deficient strain of Escherichia coli. Immediately prior to cell lysis, p51 cell paste was mixed with cell paste containing the corresponding overexpressed p66 subunit in a ratio resulting in an excess of the smaller subunit with respect to the larger. During the subsequent chromatography steps stable heterodimer p66/p51 was purified to homogeneity. This protein was characterized by amino acid analysis, denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis, analytical gel filtration HPLC, laser desorption mass spectroscopy, and isoelectric focusing. In addition, we were able to obtain crystals of the purified enzyme complexed with a quinazolinone class nonnucleoside inhibitor that diffracted to 3.2 A resolution. A potential application of this expression/purification methodology is the ability to alter specific amino acids residues, by site-directed-mutagenesis, of only one subunit of the RT-dimer.

Inhibition of HIV-1 reverse transcriptase by a quinazolinone and comparison with inhibition by pyridinones. Differences in the rates of inhibitor binding and in synergistic inhibition with nucleoside analogs.

6-Chloro-(4S)-cyclopropyl-3,4-dihydro-4-((2-pyridyl)-ethynyl)quinazol in- 2(1H)-one (L-738,372) is representative of a novel structural class of nonnucleoside inhibitors of human immunodeficiency virus, strain 1 (HIV-1), reverse transcriptase (RT), the quinazolinones. L-738,372 is a reversible inhibitor of HIV-1 RT and is noncompetitive against dTTP with a Ki of 140 nM with poly(rA).oligo(dT) as primer-template. Mixed noncompetitive inhibition by L-738,372 was observed against poly(rC).oligo(dG) as primer-template. This quinazolinone binds to RT at a site that overlaps the binding site of other nonnucleoside inhibitors as evidenced by the ability of L-738,372 to displace bound radiolabeled L-696,229, a member of the pyridinone class of inhibitors of HIV-1 RT, from complexes of RT and primer-template. Inhibition by L-738,372 shows slow binding characteristics in reactions with all of the primer-templates employed. Synergistic inhibition of RT activity was evident in combinations of L-738,372 and any of the nucleoside analogs, azidothymidine triphosphate, dideoxyinosine triphosphate, or dideoxycytosine triphosphate. The azidothymidine-resistant form of RT (D67N, K70R, T215Y, K219Q) is inhibited by L-738,372 with 2-3-fold more potency than is the wild-type RT. Comparison of inhibition by L-738,372 with inhibition by pyridinone inhibitors reveals differences in synergistic inhibition with nucleoside analogs and in the rates of binding of the inhibitors.

Sensitivity of HIV-1 reverse transcriptase and its mutants to inhibition by azidothymidine triphosphate.

HIV-1 reverse transcriptase can catalyze the addition of either azidothymidine monophosphate (AZTMP) or thymidine monophosphate (dTMP) to a primer strand opposite template adenosine bases. The ratio of incorporation of AZTMP to dTMP as catalyzed by HIV-1 reverse transcriptase has been determined to be 0.4 using an RNA-DNA duplex substrate prepared from oligonucleotides with sequences taken from the HIV-1 genome sequence. Slight variations are found for the incorporation ratio of the two nucleotides on other substrates. Substrates containing more than one adenosine in the single-stranded part of the template allow for more chances to incorporate AZTMP and less full-length product. Variations in the intensity of bands on an autoradiograph of a DNA sequencing gel corresponding to different positions of incorporation of AZTMP suggest that not all template adenosine positions offer the same level of discrimination against incorporation of AZTMP. A reverse transcriptase containing a set of four mutations (D67N, K70R, T215Y, K219Q) known to cause resistance to AZT in cell culture assays has a ratio of incorporation that is 0.77 +/- 0.03 times the ratio for the wild-type reverse transcriptase opposite one specific template adenosine. In contrast, a hybrid mutant containing the same four mutations that cause resistance to AZT and an additional mutation, Y181C, which by itself causes resistance to the non-nucleoside inhibitor L-697,661 [Sardana et al. (1992), J. Biol. Chem. 267, 17526-17530], has a ratio of incorporation that is 1.34 +/- 0.01 times that of the wild-type, indicating that the hybrid mutant enzyme is more susceptible to inhibition by AZTTP than the wild-type reverse transcriptase.(ABSTRACT TRUNCATED AT 250 WORDS)

Antitumor effect of deferoxamine on human hepatocellular carcinoma growing in athymic nude mice.

BACKGROUND: Iron is essential for the growth of all living cells. One of the important intracellular roles for iron is in the activation of ribonucleotide reductase, the enzyme that catalyzes the first step in DNA synthesis. Thus, the intracellular iron level may serve as a regulator of cell growth. The authors tested the hypothesis that lowering body iron concentration inhibits the growth of human-derived hepatocellular carcinoma (HCC) cells by depleting these cells of iron. Deferoxamine (DFO), an iron-chelating agent, was used to lower intracellular iron level. METHODS: HCC cells, PLC/PRF/5 (7 x 10(6) cells/mouse), were transplanted subcutaneously into athymic nude mice. When tumors reached 200-300 microliters in size, mice with comparable tumor sizes were paired; one was treated with DFO (300 mg/kg body weight/day, 5 days/week) intraperitoneally while the other received no treatment. RESULTS: Eight pairs of mice with HCC were observed for 5-18 weeks. Mean tumor growth rates (TGR) (mean +/- standard error) for the untreated and treated mice were 30.5 +/- 3.7 microliters/week and 11.9 +/- 1.5 microliters/week. The difference was significant (P < 0.02). In the second set of studies, DFO treatment was begun when the tumor size was smaller (100-200 microliters). Four pairs of mice were observed for 4-15 weeks; mean TGR for the four untreated mice was 18.1 +/- 5.1 microliters/week. In two mice treated with DFO, tumors regressed completely by the seventh week after initiation of treatment. The two remaining mice on DFO therapy had much slower growing tumors, with a mean TGR of 1.8 +/- 0.5 microliters/week. CONCLUSIONS: Thus, our results suggest that (1) reduction of intracellular iron concentration by DFO may be useful as antitumor therapy in HCC and (2) the favorable effects of DFO treatment are best seen when treatment is begun when the tumor is small.

Iron enhances tumor growth. Observation on spontaneous mammary tumors in mice.

Iron is essential for the growth of all cells, including tumor cells. The authors previously reported that a variety of transplantable tumors grew faster and larger in mice that were on an iron-rich diet compared with those on an iron-deficient diet. In this study the authors examined the relationship between iron in the diet and development of tumors in mice that are known to develop spontaneous tumors--C3H/HeN-MTV+(C3H-MTV+) mice that were congenitally infected with mammary tumor virus. These mice have a greater than 96% chance of developing mammary tumors between the ages of 7.2 and 9.2 months. Fifteen C3H-MTV+ weanlings were given a low-iron diet (5 mg iron/kg diet), and 15 were given diets with normal amounts of iron (180 mg Fe/kg diet). Thirteen of the 15 mice from the low-iron group and all 15 mice from the normal-iron group developed tumors. The average tumor growth rate in the normal-iron group was 112%/wk, compared with 62%/wk for the low-iron group. The difference in tumor growth rate between the two groups was significant (P = 0.02 by Student's t test). In this study, low iron intake did not prevent tumor development, but the results confirm the authors' previous report that iron nutrition of the host affects tumor growth; tumors grow better in an iron-rich environment. High levels of iron in the diet may enhance tumor growth, and this should be considered when treating patients with cancer.

Prognostic importance of serum transferrin and ferritin in childhood Hodgkin's disease.

The relationship of iron-binding proteins to prognosis was studied in 50 children at the Children's Hospital of Philadelphia, newly diagnosed with Hodgkin's disease (HD). There were five patients with Stage I, 18 with Stage II, 14 with Stage III, and 13 with Stage IV. Initial serum ferritin, transferrin, iron, hemoglobin (Hb), erythrocyte sedimentation rate (ESR), and A or B symptoms were analyzed for their association with progression-free survival (PFS). There was a linear increase of mean and median ferritin levels and a decrease of mean and median transferrin levels with advancing stages. Also, there was a significant inverse correlation between ferritin and transferrin (P less than 0.001). In univariate analyses, high ferritin (greater than 142 ng/ml) (P = 0.02) and low transferrin (less than or equal to 250 mg/dl) (P = 0.008) were significantly associated with poor PFS. Serum iron, Hb, ESR, and A or B symptoms were not associated with PFS. Stepwise proportional hazards regression analysis of all factors showed that transferrin was the only factor significantly associated with PFS. These preliminary results suggest that serum transferrin can also be used as a prognostic factor in addition to serum ferritin and that it may be helpful to assay both serum ferritin and transferrin as prognostic factors in childhood HD. Further testing of large groups of patients is needed to determine whether they are independent of tumor bulk and other established prognostic factors.

Effect of iron and desferoxamine on cell growth and in vitro ferritin synthesis in human hepatoma cell lines.

To investigate the effects of iron supplementation on hepatoma cell growth, cells from a human hepatoma cell line, PLC/PRF/5, were grown in RPMI 1640 supplemented with 0, 10 and 20 micrograms/ml of FeSO4 and harvested weekly. At the end of 6 wk culture, cell mass measured 9.6, 14.7 and 13.2 gm, respectively. Amounts of ferritin from these cell masses were 0 (undetectable), 0.89 and 2.27 micrograms/gm of cells. To study the effects of iron deprivation of hepatoma cells, three human hepatoma cell lines (PLC/PRF/5, Hep G2 and Hep 3B) were incubated in tissue culture medium mixed with graded amounts of an iron-chelating agent, desferoxamine, for 48 to 96 hr at 37 degrees C with 5% CO2. Over 50% cell death in PLC/PRF/5 cells and 30% to 50% cell death in Hep G2 and Hep 3B cells were observed 48 to 72 hr after exposure to desferoxamine. Addition of ferric citrate partially reversed the cytotoxic effect of desferoxamine. On the other hand, viability of control cells, human diploid cell line (WI 38), was not affected by desferoxamine. Even after 96 hr exposure to desferoxamine, cell death was only 2% to 4%. These results suggest that (a) iron enhances tumor cell growth, (b) iron induces increased ferritin synthesis by tumor cells in vitro and (c) iron depletion causes tumor cell death but has little effect on normal human diploid cells. These findings should be considered when designing treatment of patients with hepatoma. Iron oversupply in patients with cancer might enhance tumor growth and adversely affect cancer therapy. Iron chelation with desferoxamine might have a place in the treatment of patients with hepatoma in conjunction with other anticancer agents.

Effects of isoferritins on human granulocytes.

Serum ferritin levels are often elevated in patients with certain cancers and these elevations are, in part, derived from the tumors. In such patients, the increased levels of serum ferritin are associated with a poor prognosis. This association may be explained in part by biological effects of ferritin on lymphocytes: inhibition of E-rosette formation, masking of cell surfaces and suppression of lymphocytes' response to mitogens in vitro. The authors hypothesized that ferritins from tumor tissues also exert adverse effects on human granulocytes that are involved in tumoricidal activity. Three granulocyte functions were tested: nitroblue tetrazolium test, phagocytosis, and production of hydrogen peroxide. The results supported the authors' hypothesis: NBT reduction and phagocytosis are decreased in granulocytes exposed to ferritins, more so with tumor ferritins, than normal ferritin, and H2O2 production is less in granulocytes previously exposed to ferritins from tumor and nontumor tissues than cells not exposed to ferritins. However, the inhibitory effects of ferritins on H2O2 production can be reversed if granulocytes are further stimulated by phorbol myristate acetate (a membrane stimulant). If the elevated serum ferritin in cancer patients impairs granulocyte functions, in vivo, then it may increase the risk of infection, decrease tumoricidal host responses, and, thereby, contribute to the poor prognosis of these individuals.