Cyclophosphamide followed by fludarabine for untreated chronic lymphocytic leukemia: a phase II SWOG TRIAL 9706.

B-cell chronic lymphocytic leukemia (CLL) accounts for 95% of chronic leukemia cases and 25% of all leukemia. Despite the prevalence of CLL, progress in its treatment has been only modest over the past three decades. Based upon the ability of fludarabine to produce high-grade remissions especially among patients with low initial tumor mass, and the ability of alkylators to reduce tumor mass, we hypothesized that sequential administration of a limited number of cycles of intermediate-dose cyclophosphamide followed by fludarabine could result in a larger percentage of patients with complete remissions (CRs). In all, 27 of the 49 eligible patients achieved overall responses of CR, unconfirmed complete remission (UCR), or PR, for a total response rate of 55% (95% confidence interval (CI) 40-69%). Considering the confounding medical issues of this patient population with advanced aggressive disease, the regimen was generally well tolerated. This study demonstrates that high-dose cyclophosphamide followed by fludarabine was relatively well tolerated in this group of advanced CLL patients. The study's criterion for testing whether the regimen is sufficiently effective to warrant further investigation was met: 14 (32%) of the first 44 eligible patients achieved CR or UCR.

Expression of different NF-kappaB pathway genes in dendritic cells (DCs) or macrophages assessed by gene expression profiling.

NF-kappaB/Rel transcription factors have been implicated in the differentiation of monocytes to either dendritic cells (DCs) or macrophages, as well as in the maturation of DCs from antigen-processing to antigen-presenting cells. Recent studies of the expression pattern of Rel proteins and their inhibitors (IkappaBs) suggest that their regulation during this differentiation process is transcriptional. To investigate differential gene expression between macrophages and DCs, we used commercially available gene microarrays (GEArray KIT), which included four of the NF-kappaB/Rel family genes (p50/p105, p52/p100, RelB, and c-rel) and 32 additional genes either in the NF-kappaB signal transduction pathway or under transcriptional control of NF-kappaB/Rel factors. To generate macrophages and DCs, human adherent peripheral blood monocytes were cultured with M-CSF or GM-CSF + IL-4 respectively for up to 8 days. DCs (and in some experiments, macrophages) were treated with lipopolysaccharide (LPS) for the last 48 h of culture to induce maturation. Cells were harvested after 7 days, cDNA was prepared and radiolabeled with alpha-(32)P-dCTP, then hybridized to gene arrays containing specific gene probes. beta-actin and GAPDH or PUC18 oligonucleotides served as positive or negative controls, respectively. The expression of all four NF-kappaB/Rel family genes examined was significantly upregulated in maturing DCs compared to macrophages. The strongest difference was observed for c-rel. RT-PCR determinations of c-rel, RelB, and p105 mRNAs confirmed these observations. Among the 32 NF-kappaB/Rel pathway genes, 14 were upregulated in mature DCs compared to macrophages. These genes were IkappaBalpha, IKK-beta, NIK, ICAM-1, P-selectin, E-selectin, TNF-alpha, TNFR2, TNFAIP3, IL-1alpha, IL-1R1, IL-1R2, IRAK, and TANK. By contrast, only mcp-1 (monocyte chemotactic protein 1) was upregulated in macrophages compared to DCs. NF-kappaB pathway genes upregulated in DCs compared to macrophages were constitutively expressed in monocytes then selectively downregulated during macrophage but not DC differentiation. LPS did not induce expression of most of these genes in macrophages but LPS did induce upregulation of IL-8 in mature macrophages. We conclude that NF-kappaB/Rel family genes, especially c-rel, are selectively expressed during differentiation of monocytes towards DCs. Moreover, this differential expression is associated both with activation of different NF-kappaB signal transduction pathways in DCs and macrophages and with expression of a unique subset of genes in DCs that are transcriptionally targeted by NF-kappaB/Rel factors. The results illustrate the ability of the NF-kappaB pathway to respond to differentiation stimuli by activating in a cell-specific manner unique signalling pathways and subsets of NF-kappaB target genes.

Expression of multiple genes regulating cell cycle and apoptosis in differentiating hematopoietic cells is dependent on iron.

OBJECTIVE: Iron plays critical roles in many biological processes including hematopoietic cell growth and differentiation. Iron is essential for the differentiation of HL-60 promonocytes. HL-60 cells stimulated with phorbol myristate acetate (PMA) undergo G1/S phase cell-cycle arrest and differentiate to monocyte/macrophages. With iron deprivation, PMA-induced HL-60 cells bypass differentiation and undergo apoptosis. To investigate the molecular basis underlying this observation, we used commercially available gene microarrays to evaluate expression of multiple genes involved in the regulation of cell cycling and apoptosis. METHODS: We treated HL-60 cells with PMA +/- desferrioxamine (DF), a potent iron chelator, to produce iron deprivation. Cells were cultured for 48 hours, and cDNA was prepared and radiolabeled with alpha-(32)P dCTP, then hybridized to gene arrays containing specific cDNA fragments. RESULTS: Expression of 11 of 43 genes was inhibited greater than 50% by iron deprivation. These genes were Rb; p21 (WAF1/CIP1); bad; cdk2; cyclins A, D3, E1; c-myc; egr-1; iNOS; and FasL. For each gene the microarray results were confirmed by RT-PCR and/or Northern or Western blotting. Nuclear transcription assays indicated that the role of iron in Rb expression was to support gene transcription. Addition of ferrioxamine (iron saturated DF) instead of DF to PMA-induced cells did not affect gene expression, indicating that diminished expression was due to iron deprivation, not nonspecific toxicity. CONCLUSION: Iron supports expression of multiple cell cycle-regulatory and apoptosis-related genes during HL-60 cell differentiation, and, in this way, is involved in regulation of a critical cell decision point-the decision to pursue a differentiation-related or apoptotic pathway.

Comparison of the L10M consolidation regimen to an alternative regimen including escalating methotrexate/L-asparaginase for adult acute lymphoblastic leukemia: a Southwest Oncology Group Study.

The effectiveness of intensive post-remission chemotherapy regimens for adult patients with acute lymphoblastic leukemia (ALL) is limited by both a high rate of disease recurrence and a substantial incidence of treatment toxicity. To evaluate a potentially more effective and less toxic approach, we conducted a multicenter phase III trial of consolidation therapies comparing the standard L10M regimen with one combining the brief, intensive L17M regimen and escalating methotrexate (MTX) and L-asparaginase (L-asp). Patients over age 15 with previously untreated ALL were eligible. Induction therapy included vincristine, prednisone, doxorubicin, cyclophosphamide and intrathecal methotrexate administered over 36 days. Patients who achieved complete remission (CR) were randomized to receive consolidation with either the L10M regimen or with DAT (daunomycin, cytosine arabinoside, 6-thioguanine) and escalating MTX and L-asp. The randomization was stratified by age, WBC and Ph chromosome status. Maintenance therapy was the same in both arms. Of 353 eligible patients, 218 (62%) achieved CR and 195 were randomized. The treatment arms did not differ significantly with respect to disease-free survival (DFS; P= 0.46) or overall survival (P= 0.39). Estimated DFS at 5 years was 32% (95% confidence interval (CI) 23-42%) in the L10M arm and 25% (95% CI 16-33%) in the DAT/MTX/L-asp arm. In each arm, 4% of patients died of toxicities (infection in all but one case). Infections and nausea/vomiting were somewhat more common in the L10M arm (occurring in 68% and 53% of patients respectively) than the DAT/MTX/L-asp arm (56% and 33%). The DAT/MTX/L-asp consolidation regimen was associated with some reduction in nonfatal toxicities, but no significant improvement in DFS, overall survival or non-relapse mortality when compared to the standard L10M regimen.

A new molecular role for iron in regulation of cell cycling and differentiation of HL-60 human leukemia cells: iron is required for transcription of p21(WAF1/CIP1) in cells induced by phorbol myristate acetate.

To investigate the role of iron in hematopoiesis, we studied effects of iron deprivation on PMA-induced monocyte/macrophage differentiation in HL-60 cells. Iron deprivation induced by desferrioxamine (DF) blocked PMA-induced differentiation and induced S-phase arrest and apoptosis in up to 60% of cells. Apoptosis was not related to a decrease of bcl-2 or to c-myc overexpression. In the presence of DF, PMA-induced upregulation of the cyclin dependent kinase inhibitor (CDKI), p21(WAF1/CIP1), was blocked and its expression could be restored in the presence of DF by supplementation with ferric citrate. Furthermore, ferrioxamine (iron saturated DF) did not block induction of p21(WAF1/CIP1) indicating that the changes were not due to a nonspecific toxic effect of DF. Similarly, hydroxyurea, an inhibitor of ribonucleotide reductase, did not block p21 expression. p21(WAF1/CIP1) antisense oligonucleotides caused cell cycle alterations similar to DF and p21 overexpression overcame effects of iron deprivation on both cell cycling and differentiation. Therefore, p21 is a key target for the effects of iron deprivation on HL-60 cell cycling and differentiation. Nuclear run-on transcription assays and p21 mRNA half-life studies indicated that iron was required to support transcriptional activation of p21(WAF1/CIP1) after a PMA stimulus. By contrast, iron deprivation did not inhibit expression of a second CDKI, p27(KIP1). These data demonstrate a new role for iron during monocyte/macrophage differentiation. A key role of iron is to allow induction of p21(WAF1/CIP1) in response to a differentiation stimulus subsequently blocking cells at the G(1)/S cell cycle interface and preventing premature apoptosis. This effect of iron is independent of its requirement in supporting the activity of the enzyme, ribonucleotide reductase. Because of the central role of p21(WAF1/CIP1) as regulator of the G(1)/S cell cycle checkpoint this requirement for iron to support p21 expression represents an important mechanism by which iron may modulate hematopoietic cell growth and differentiation. Published 2001 Wiley-Liss, Inc.

A phase II study of high dose ARA-C and mitoxantrone for treatment of relapsed or refractory adult acute lymphoblastic leukemia.

PURPOSE: The Southwest Oncology Group performed a Phase II study to investigate the effectiveness of an induction regimen of high dose cytosine arabinoside (ara-C) with high dose mitoxantrone for treatment of relapsed or refractory adult acute lymphoblastic leukemia (ALL). PATIENTS AND METHODS: Patients at least 16-years-old with ALL that was in relapse after, or was refractory to, standard induction therapy including at least vincristine and prednisone were eligible, as long as they had no prior treatment with high dose ara-C. The induction regimen included high dose ara-C (3 g/m2 by 3-h i.v. days 1-5) and mitoxantrone (80 mg/m2 by 15-30 min i.v. 12-20 h after the first dose of ara-C). The study design called for a maximum of 55 patients, with early termination if less than nine of the first 30 achieved complete remission. RESULTS: Thirty-three patients entered the study, and 31 were included in the analysis. All 31 completed one course of induction therapy. Four patients died of infection and a fifth of cardiomyopathy with possible sepsis. Seven patients achieved complete remission (23%; 95% confidence interval 10-41%). One of the seven received syngeneic bone marrow transplantation while in remission, and the other six all relapsed within 10 months. All 31 patients died within 25 months after entering the study. CONCLUSIONS: The regimen of high dose ara-C and mitoxantrone was found to be insufficiently effective to warrant further investigation.

Cell cycle regulatory gene abnormalities are important determinants of leukemogenesis and disease biology in adult acute lymphoblastic leukemia.

To test the hypothesis that cell cycle regulatory gene abnormalities are determinants of clinical outcome in adult acute lymphoblastic leukemia (ALL), we screened lymphoblasts from patients on a Southwest Oncology Group protocol for abnormalities of the genes, retinoblastoma (Rb), p53, p15(INK4B), and p16(INK4A). Aberrant expression occurred in 33 (85%) patients in the following frequencies: Rb, 51%; p16(INK4A), 41%; p53, 26%. Thirteen patients (33%) had abnormalities in 2 or more genes. Outcomes were compared in patients with 0 to 1 abnormality versus patients with multiple abnormalities. The 2 groups did not differ in a large number of clinical and laboratory characteristics. The CR rates for patients with 0 to 1 and multiple abnormalities were similar (69% and 54%, respectively). Patients with 0 to 1 abnormality had a median survival time of 25 months (n = 26; 95% CI, 13-46 months) versus 8 months (n = 13; 95% CI, 4-12 months) for those with multiple abnormalities (P <.01). Stem cells (CD34+lin-) were isolated from adult ALL bone marrows and tested for p16(INK4A) expression by immunocytochemistry. In 3 of 5 patients lymphoblasts and sorted stem cells lacked p16(INK4A) expression. In 2 other patients only 50% of sorted stem cells expressed p16(INK4A). By contrast, p16 expression was present in the CD34+ lin- compartment in 95% (median) of 9 patients whose lymphoblasts expressed p16(INK4A). Therefore, cell cycle regulatory gene abnormalities are frequently present in adult ALL lymphoblasts, and they may be important determinants of disease outcome. The presence of these abnormalities in the stem compartment suggests that they contribute to leukemogenesis. Eradication of the stem cell subset harboring these abnormalities may be important to achieve cure.

New perspectives on iron: an introduction.

Iron is an essential nutritional element for all life forms. Iron plays critical roles in electron transport and cellular respiration, cell proliferation and differentiation, and regulation of gene expression. Two emerging new functions for iron are its necessary role in supporting transcription of certain key genes required for cell growth and function [eg, nitric oxide synthase, protein kinase C-beta, p21 (CIP1/WAF1)] and its complex role in hematopoietic cell differentiation. However, iron is also potentially deleterious. Reactive oxygen species generated by Fenton chemistry may contribute to major pathological processes such as cancer, atherosclerosis, and neurodegenerative diseases. Iron-generated reactive oxygen species may also function in normal intracellular signaling. Therefore, roles of iron are both essential and extraordinarily diverse. This symposium explores this diversity by covering topics of iron absorption and transport, the regulation of gene expression by iron responsive proteins, the cellular biology of heme, hereditary hemochromatosis, and clinical use of serum transferrin receptor measurements.

Ig heavy chain third complementarity determining regions (H CDR3s) after stem cell transplantation do not resemble the developing human fetal H CDR3s in size distribution and Ig gene utilization.

Previous studies have suggested that the B-cell repertoire after stem cell transplantation resembles the developing repertoire in the fetus. Fetal and adult repertoires differ strikingly at the molecular level in Ig heavy chain third complementarity determining region (H CDR3) size distribution and Ig gene utilization. Previously, the posttransplant repertoire has not been studied fully in this regard. In this study, we analyzed H CDR3s posttransplant using CDR3 fingerprinting, single-strand conformation polymorphism (SSCP), and random sequencing. Eleven adult patients who received either autologous (n = 6) or allogeneic adult sibling (n = 5) hematopoietic stem cell transplants were studied. IgM H CDR3 repertoires demonstrated limited clonal diversity within the first 6 to 10 weeks posttransplant. By 3 to 4 months, the IgM H CDR3 repertoires were as diverse as those in healthy adults. Reconstitution of the IgM diversity correlated with the expansion of the multimember VH3 family. By contrast, the contribution of the single-member VH6 family was limited in most patients up to 6 to 9 months. No evidence was seen for greater contribution of VH6 posttransplant. IgG repertoires remained clonally restricted at all times. In all patients, H CDR3 sizes fell within adult limits. Direct nucleotide sequencing of H CDR3s showed adult-type N-nucleotide insertions and Ig gene utilization. These results indicate that the emerging repertoire posttransplant does not resemble the developing fetal repertoire at the molecular level.

Analysis of DNA binding proteins associated with hemin-induced transcriptional inhibition. The hemin response element binding protein is a heterogeneous complex that includes the Ku protein.

Hemin inhibits transcription of the tartrate resistant acid phosphatase (TRAP) gene. Using deletion mutagenesis of the mouse TRAP 5'-flanking region, we previously identified a 27-bp DNA segment containing a central GAGGC tandem repeat sequence (the hemin response element [HRE]), which bound nuclear proteins (hemin response element binding proteins [HREBPs]) from hemin-treated cells and appeared to be responsible for mediating transcriptional inhibition in response to hemin. We now have used affinity binding to HRE-derivatized beads to identify four HREBP components with apparent molecular masses of 133-, 90-, 80-, and 37-kD, respectively. The 80- and 90-kD components correspond to the p70 and p80/86 subunits of Ku antigen (KuAg) as documented by partial amino acid microsequencing of tryptic digests and immunologic reactivity. Based on reactivity of the HREBP gel shift band with antibodies to the redox factor protein (ref1) in shift Western experiments, it is shown that the 37-kD component represents ref1. The 133-kD component appeared to be a unique protein. KuAg participation in HREBP complexes was specific as it was present in HREBPs bound to HRE microcircles. Results of depletion/reconstitution experiments suggested that KuAg does not bind alone or directly to HRE DNA, but does so only in conjunction with the 133- and/or 37-kD proteins. We conclude that HREBP is a heterogeneous complex composed of KuAg, ref1, and a unique 133-kD protein. We speculate that the role of heme may be to promote interactions among these components, thereby facilitating HRE binding and downregulation of hemin responsive genes.

Tumor suppressor gene alteration in adult acute lymphoblastic leukemia (ALL). Analysis of retinoblastoma (Rb) and p53 gene expression in lymphoblasts of patients with de novo, relapsed, or refractory ALL treated in Southwest Oncology Group studies.

To examine the impact of inactivation of tumor suppressor genes on outcome in adult ALL, we compared two groups of patients registered to SWOG treatment protocols for loss of the Rb gene product and p53 overexpression: (1) 89 patients with de novo ALL, and (2) 26 patients with relapsed/refractory ALL. The groups were comparable with respect to age, sex, and race. Cell lysates (> or = 80% blasts) were analyzed by immunoblotting which enabled detection of Rb or p53 proteins in as little as 1 microg of lysate. Loss of Rb expression (pRbneg) was found in 54/85 (64%) de novo and 11/19 (58%) relapsed patients (P = 0.79). Overexpression of p53 (p53abn), indicative of p53 point mutations, was found in 16/75 (21%) de novo and 8/19 (42%) relapsed patients (P = 0.08). Using a nonisotopic RNase cleavage assay, p53 point mutations in exons 5-9 were confirmed in 14/23 (61%) p53abn specimens. For the de novo ALL group, patients with normal Rb protein had higher WBC and higher peripheral blast and lymphocyte counts. Otherwise neither abnormal Rb or p53 expression correlated with any of a large panel of clinical and laboratory variables including FAB class, blast lineage, expression of myeloid antigens or CD34, and presence of the Ph1 chromosome or BCR-ABL. Analyses of treatment outcomes demonstrated no significant impact of Rb or p53 status alone on CR rates, relapse-free or overall survival. An identical percentage (11%) of both de novo and relapsed/refractory patients had concurrent abnormalities of both Rb and p53 expression (pRbneg/p53abn). The survival curve of these patients suggests an increased rate of early death, but the number of patients in this group was small. Summarizing, (1) loss of Rb expression is common in adult ALL; (2) overexpression of p53 may be more frequent in relapsed/refractory than de novo adult ALL; and (3) although Rb or p53 alterations alone are not strong independent predictors of outcome, their concurrent expression may predict a poor response to therapy.

Paediatric tracheobronchomalacia and its assessment by tracheobronchography.

Tracheobronchomalacia (TBM) is a rare condition that results in abnormal compliance of the airways with airway collapse being most marked in expiration. In a series of 28 patients, it was observed that a majority of cases presented with malacia involving the trachea (64%) and the left main bronchus (64%) alone. The right main bronchus was involved in combination with the trachea in 32%, but rarely was involved by itself. Most patients demonstrated associated congenital abnormalities, such as tracheo-oesophageal fistulae, vascular rings or congenital heart disease (78%). A small proportion were premature infants with no obvious associated abnormality (11%). Evaluation of the dynamic process, tracheobronchomalacia; requires 'real-time' investigation. While computed tomography (CT) and magnetic resonance imaging (MRI) have their role in the management of chronic airway obstruction in the child, only cine-CT, bronchoscopy, fluoroscopy and tracheobronchography provide real-time assessment for the evaluation of a dynamic process such as TBM. In our experience, tracheobronchography provides excellent anatomic, physiologic and therapeutic information in the assessment of those infants with primary or secondary TBM. By using continuous positive airway pressure in incremental doses, the amount of positive pressure required to maintain an open airway in expiration is a valuable adjunct to the clinical management of the patient. When careful attention is paid to technique, tracheobronchography is a safe investigation in experienced hands.

Inhibition of tartrate-resistant acid phosphatase gene expression by hemin and protoporphyrin IX. Identification of a hemin-responsive inhibitor of transcription.

Tartrate-resistant acid phosphatase (TRAP) is an iron-containing protein encoded by the same gene that codes for uteroferrin, a placental iron transport protein. In human peripheral mononuclear cells, TRAP expression is inhibited by both hemin (ferric protoporphyrin IX) and protoporphyrin IX. Nuclear run-on assays confirmed that this inhibition occurs at the level of gene transcription. Previous studies with mTRAP deletion mutants showed that the hemin effect was dependent on repressor activity in the mTRAP 5'-flanking region at -1846 bp to -1240 bp relative to ATG (Reddy et al, J Bone Mineral Res 10:601, 1995). We now report that gel shift assays showed a DNA binding protein in nuclear extracts of hemin-treated cells termed hemin response element binding protein (HREBP). Additional studies have localized the HREBP binding region in the mTRAP 5'-flanking DNA to a 27-bp sequence at -1815 to -1789 bp relative to ATG. A tandem repeat sequence, GAGGC;GAGGC, contained within this DNA segment, was shown to be involved in binding of HREBP. Highly homologous sequences are present in the 5'-flanking region of the hTRAP gene. Binding of HREBP to the mTRAP DNA sequence was inhibited by anti-HAP1 antibodies, indicating homology between the hemin-responsive factor and the yeast heme-dependent transcription factor, HAP1. A 607-bp segment of the mTRAP 5'-flanking region containing the candidate hemin response element and surrounding sequences conferred hemin regulation on the viral SV40 promoter. Southwestern blotting experiments probing nuclear extracts of hemin-treated U937 cells with the 27-bp binding sequence showed two protein bands at 37 and 133 kD representing candidate HREBPs. A GENINFO search showed several other mammalian genes with tandem GAGGC motifs in noncoding regions, providing the possibility that additional genes may also be regulated by hemin at the level of transcription. These studies provide the first description of a novel iron/hemin-responsive transcriptional regulatory mechanism in mammalian cells.

Sinusitis and intracranial sepsis: the CT imaging and clinical presentation.

The CT imaging and clinical presentation in 14 children with coexistent intracranial sepsis and sinusitis were reviewed. A routine CT head scan (10-mm thick semi-axial slices through the cranium done before and after intravenous contrast medium administration) was found to be an inadequate initial investigation as the intracranial collection was missed in four patients and the abnormal sinuses not shown in six. In half the children the diagnosis of sinusitis was unsuspected at the time of admission. The dominant clinical features were fever, intense headache and facial swelling in early adolescent males. In this clinical setting we recommend: (1) the routine scan is extended through the frontal and ethmoidal sinuses and photographed at a window level and width showing both bone detail and air/soft tissue interfaces; (2) direct coronal projections are performed through the anterior cranial fossa if no collection is seen on the routine study; (3) an early repeat scan within 48 h if the initial study shows no intracranial pathology but the fronto-ethmoidal sinuses are abnormal and there is a high clinical suspicion of intracranial sepsis; and (4) in the presence of intracranial sepsis the vault is viewed at bone window settings to exclude cranial osteomyelitis.

Deoxyspergualin: phase I clinical, immunologic and pharmacokinetic study.

Deoxyspergualin (DSG) is an analog of the polyamine spergualin with preclinical evidence of activity in murine and human tumor models. This phase I study examined a 120 h continuous infusion schedule in 56 patients with refractory solid tumors at doses ranging from 80 to 2792 mg/m2/day. Dose-limiting toxicity was reversible hypotension and appeared to be associated with plasma levels of DSG > 4 micrograms/ml. Other dose-dependent effects noted were pruritus and circumoral paresthesias. Myelosuppression and gastrointestinal toxicities were mild and sporadic. Two patients with refractory head and neck cancer had minor responses. The recommended phase II dose on this schedule is 1800 mg/m2. Additional monitoring to identify immunologic properties included immunophenotyping of peripheral lymphocytes and cytotoxic activity by means of standard 51Cr-release assays. These studies revealed a non-dose-dependent increase in the number of cells expressing T cell antigens predominantly the T suppressor (CD8) phenotype posttreatment. In three patients, a mild increase in LAK activity was noted post-treatment without a consistent relationship to dose or change in cell surface antigens. Pharmacokinetic studies were completed on 26 patients ranging from doses of 80 to 2792 mg/m2. The average plasma concentration ranged from 0.07 to 7 micrograms/ml. DSG was rapidly cleared from the plasma with a mean terminal half-life of 1.9 h. Mean total body clearance was 25.24 l/h/m2. Further in vivo immunologic studies should be pursued while the agent is studied in fixed dosage phase II clinical trials.

Characterization of the mouse tartrate-resistant acid phosphatase (TRAP) gene promoter.

Tartrate-resistant acid phosphatase (TRAP) is an iron-binding protein that is highly expressed in osteoclasts. To characterize the regulation of TRAP gene expression, progressive 5' and 3' deletions of a 1.8 kb fragment containing the 5'-flanking sequence were fused to a luciferase reporter gene. Two nonoverlapping regions of this 1.8 kb fragment had promoter activity. The upstream promoter (P1) was located within the region from -881 bp to -463 bp relative to the ATG, while the downstream promoter (P2) was located between -363 bp to -1 bp in a region we have previously shown to be an intron in transcripts originating from the upstream promoter. A putative repressor region for the P2 promoter at -1846 bp to -1240 bp and a putative enhancer region at -962 bp to -881 bp relative to the ATG were identified. PCR analysis of promoter-specific transcription of the TRAP gene in various murine tissues showed that both promoters were active in several tissues. Transferrin-bound iron increased P1 promoter activity 2.5-fold and hemin decreased P1 promoter activity, but neither had any effect on P2 activity. These data show that the transcriptional regulation of the TRAP gene is complex and that iron may play a key role in TRAP gene regulation.

Expression of myeloid antigens by blast cells in acute lymphoblastic leukemia of adults. The Southwest Oncology Group experience.

A subset of adult acute lymphoblastic leukemia (ALL) patients have blast cells which co-express myeloid-associated antigens (MY+ ALL). We have analyzed 113 adult ALL cases for expression of MY-associated antigens (MAA). ALL was diagnosed by standard morphology, cytochemistry, and immunophenotype in central review. MY+ ALL was diagnosed when > or = 20% of lymphoblasts co-expressed CD13 and/or CD33. Overall incidence of MY+ was 31/113 (27%). MAA expression was not significantly correlated with WBC, blast count, hemoglobin, or hematocrit. MY+ cases were more likely to express B-associated antigens, especially CALLA, and to be FAB L2, Ph+, or to have the BCR-ABL translocation by PCR, but these differences were not statistically significant. All patients were induced with a L10M regimen, and 67 (59%) achieved CR: 43/66 (65%) of B MY neg; 14/29 (48%) of B MY+; 10/16 (63%) T MY neg; and 0/2 T MY+. In age-adjusted analyses CR rate did not differ significantly between MY+ and MY neg patients or between B- and T-cell patients. Of the 113 patients, 84 have died and the remaining 29 patients have been followed for a median of 49 months. In proportional hazards regression analyses adjusting for age and WBC, heterogeneity of survival among the four groups was statistically significant (p = 0.021), largely due to MY status. The mortality rate was 85% greater for MY+ patients compared to MY neg patients (two-tailed p = 0.013). By contrast, survival did not vary significantly between B- and T-cell patients. The data indicate that MAA expression is useful for predicting overall survival of adult patients with ALL treated in a L10M protocol. As a predictive factor MAA expression is comparable to the WBC and superior to the more standard stratification by B- or T-cell markers for this group of patients.

Regulation of protein kinase C (PKC) expression by iron: effect of different iron compounds on PKC-beta and PKC-alpha gene expression and role of the 5'-flanking region of the PKC-beta gene in the response to ferric transferrin.

We have studied effects of ferric transferrin (FeTF), ferric lactoferrin (FeLF), ferric complexes of pyridoxal- or salicylaldehyde-isonicotinoyl hydrazone, (Fe-PIH, Fe-SIH), and ferric ammonium citrate (FAC) on expression of protein kinase C (PKC) mRNA transcripts in a variety of cultured cell lines. FeTF supported an increase of PKC-beta mRNA transcripts in T-lymphoblastoid (CCRF-CEM; Jurkat), B-lymphoblastoid (Daudi; Raji), promyelocyte (HL-60), erythroleukemia (K562), and monocyte (U937) cell lines. By contrast, FeLF, Fe-PIH, and Fe-SIH did not support an increase of PKC-beta mRNA transcripts in any of these cell lines. Furthermore, FAC supported an increase of PKC-beta mRNA transcripts in HL-60, K562, and U937 cells only. Preincubation of cells with desferrioxamine (DF), a cell-permeable iron chelator, abolished the increments of PKC-beta mRNA observed in response to FeTF or FAC. In contrast to results with PKC-beta, neither FeTF nor FAC caused an increase of PKC-alpha transcripts in any cell line. To locate iron-responsive DNA regulatory elements of the PKC-beta gene, we prepared genetic constructs containing various portions of the human PKC-beta 5'-flanking DNA linked to the firefly luciferase gene. Constructs were cotransfected with the neomycin resistance plasmid, Pwl-neo, into HRE H9 cells, and stable transfectants were selected in G418. Treatment with FeTF of transfectants bearing chimeric gene constructs with 2,200 bp of the PKC-beta 5'-flanking region increased luciferase activity and mRNA transcripts 2.5-fold. This increase was blocked by DF. Neither luciferase activity nor mRNA increased with FeTF in stable transfectants bearing constructs with 342 bp or 587 bp of the PKC-beta 5'-flanking region. These data provide direct confirmation that iron is involved in regulation of PKC-beta but not PKC-alpha gene expression in many cell lines. The form in which iron is presented to these cell lines appears to affect its availability for this function, and cells vary in their capabilities to use nontransferrin iron to support PKC-beta gene expression. Finally, transcriptional upregulation of PKC-beta by FeTF is mediated by DNA sequences located between -2200 bp and -587 bp in the 5'-flanking region of the human PKC-beta gene.