Activation of EVI1 gene expression in human acute myelogenous leukemias by translocations spanning 300-400 kilobases on chromosome band 3q26.

Retroviral activation of Evi-1 gene expression is one of the most common transforming events in murine myeloid leukemias. To evaluate the role of the EVI1 gene in human acute myelogenous leukemia (AML), leukemic blasts or cell lines from 116 patients were examined. In eight patients the EVI1 gene was expressed and all but one had cytogenetically detectable translocations of chromosome 3q26 where the EVI1 gene has been localized. To identify breakpoints, a restriction map that spans 1700 kilobases (kb) of the EVI1 locus was developed by pulsed-field gel electrophoresis. In one case, t(3;3)(q21;q26), a rearrangement was localized to 170-330 kb 5' of the gene. In a second case, t(3;3)(q21;q26), there was a rearrangement 13 kb 5' of the gene. This rearrangement was cloned and shown to be due to the fusion of sequences from 3q21-22 with the EVI1 locus. In the third case, ins(3)-(q21q25q27), there was a rearrangement that mapped 150 kb downstream from the 5' end of the gene.     

Characterization of a factor-dependent acute leukemia cell line with translocation (3;3)(q21;q26).

A strictly factor-dependent cell line (UCSD/AML1) was established from a patient with the syndrome of multilineage acute leukemia with high platelets. The patient's cells and the cell line karyotype were 45,XX,-7,t(3;3)(q21;q26), typical of the syndrome of acute leukemia with high platelets. The cell line expresses CD34, CD7, TdT, and myeloid (CD13, CD14, CD33) and megakaryocyte/platelet (CD36, CD41, CD42b, CDw49b) antigens. In short-term culture, UCSD/AML1 cells proliferate in response to interleukin-3 (IL-3), IL-4, IL-6, macrophage colony-stimulating factor (M-CSF), and granulocyte-macrophage CSF (GM-CSF), but not IL-1, IL-2, IL-5, or G-CSF. In long-term culture, proliferation can be sustained by GM-CSF, IL-6, or M-CSF. When maintained in GM-CSF, a small percentage of cells form multinucleated megakaryocyte-like giant cells. Culture with GM-CSF combined with IL-6, but not with IL-6 alone, increased giant cell formation fourfold to sevenfold. IL-6 alone or in combination with GM-CSF increased expression of platelet-related antigens. In contrast, culture with phorbol ester induced formation of macrophage-like cells. UCSD/AML1 is the first human acute nonlymphocytic leukemia cell line established from a patient with an acute leukemia syndrome associated with a specific chromosome abnormality.     

Non-cross-resistant chemotherapy and consolidation radiotherapy for small cell carcinoma of the lung

Fifty-six patients with small cell carcinoma of the lung were treated with a two-cyclic induction course of hexamethylmelamine, vincristine, doxorubicin, and cyclophosphamide. Patients with limited disease (LD) who responded and patients with extensive disease (ED) who had a complete response received prophylactic whole-brain radiotherapy, as well as radiotherapy to thoracic and abdominal sites of disease. Concurrently with radiotherapy, consolidation chemotherapy was given with doxorubicin, cyclophosphamide, methotrexate, and etoposide. The complete response rate was 35% for ED patients and 68% for LD patients. The median survival time for complete responders was 54 weeks for ED patients and 65 weeks for LD patients. The toxicity of the program was moderate, and the effectiveness was comparable to that of other reported combined-modality treatment programs.     

Preclinical evaluation of illudins as anticancer agents

Illudins are low molecular weight natural products which were previously evaluated as anticancer drugs using rodent tumor models. In the present studies, we used in vitro cultures of human cancer cells to reevaluate their potential as anticancer agents. Using continuous exposure, Illudins S and M were cytotoxic to human leukemia cells at concentrations of 6-100 nM, but dihydroilludin M was 3 orders of magnitude less toxic, thus identifying a ketone site as a structural feature critical for cytotoxicity. Cytokinetic studies showed that illudin S caused a complete block at the G1-S phase interface of the cell cycle. Kinetics of inhibition of radiolabeled thymidine, uridine, and leucine incorporation suggested a primary effect on DNA synthesis. In colony and liquid culture assays, cell killing was time dependent but near maximal with a 2-h exposure. Myeloid and T-lymphocyte leukemia cells were most sensitive (50% inhibitory concentration, 6-11 nM), but B-cell leukemia/lymphoma, melanoma, and ovarian carcinoma cells were at least 10 times more resistant. Bone marrow granulocyte/macrophage progenitors showed intermediate sensitivity. Illudin S was equally effective against CEM T-lymphocyte leukemia cells expressing the multidrug resistance phenotype associated with Mr 180,000 glycoprotein and the parental cell line. CEM cells resistant to doxorubicin, epipodophyllotoxins, and 1-beta-D-arabinofuranosylcytosine showed only a 2-fold increased resistance to illudin S. Illudins are novel and potent cytotoxins which may be preferentially active against human myeloid and T-cell leukemias, including cells resistant to more conventional chemotherapeutic agents. The present studies illustrate the breadth of information which can be obtained on a new agent using present in vitro screening procedures and human cells.     

Preclinical evaluation of illudins as anticancer agents: basis for selective cytotoxicity

Illudins are potent natural products derived from Omphalotus illudens and related fungi. The chemical structure of illudins differs from that of other conventional chemotherapeutic agents. While illudins are toxic to most tumor cells after prolonged exposure (greater than or equal to 48 hr), with shorter exposure times (less than or equal to 2 hr), they show selective toxicity for human myelocytic leukemia and epidermoid, lung, ovarian, and breast carcinoma cells of various species of origin. The apparent histologic specificity of illudin S toxicity is based on an energy-dependent transport mechanism present in sensitive cells, but absent in cells relatively resistant to illudin S. For human myeloid leukemia HL60 cells, the Michaelis constant was 4.2 microM and the maximum velocity was 12.2 pmol/minute per 10 million cells or 730 pmol/hour per 10 million cells. The energy-dependent transport mechanism was detected in other mammalian tumor cells.     

Sequential cycles of high-dose carboplatin administered with recombinant human granulocyte-macrophage colony-stimulating factor and repeated infusions of autologous peripheral-blood progenitor cells: a novel and effective method for delivering multiple courses of dose-intensive therapy.

PURPOSE: The trial was undertaken to study the effect of administering granulocyte-macrophage colony-stimulating factor (GM-CSF) with and without peripheral-blood progenitor cells (PBPC) on the hematologic and nonhematologic toxicity observed with multiple cycles of high-dose carboplatin chemotherapy. PATIENTS AND METHODS: Eighteen patients with a variety of solid tumors received a total of 40 cycles of carboplatin, 1,200 mg/m2 per cycle, administered by continuous infusion over 96 hours. All 40 courses were administered with a daily 4-hour intravenous (IV) infusion of either 5 or 10 micrograms/kg/d of recombinant human Escherichia coli-derived GM-CSF. The first 20 courses were administered without PBPC support (treatment A). Because of severe neutropenia and thrombocytopenia, the next 20 courses of therapy were administered with GM-CSF, PBPC, and oral antibiotic prophylaxis (treatment B). RESULTS: The addition of PBPC support led to a significant reduction in the duration of neutropenia (10.5 v 7.5 days; P = .027) and thrombocytopenia (12.4 v 5.2 days; P = .001), number of RBC transfusions (six v three; P = .01) and platelet transfusions (10.3 v 3.7; P = .013), number of hospital days (12.6 v 2.9; P = .01), and days of IV antibiotics (11.8 v 2.4; P = .007) per cycle. Significant increases in the weekly dose intensity (206 v 285 mg/m2/wk; P = .014) and total dose (2,287 v 3,600 mg/m2; P = .018) of carboplatin delivered were also observed with treatment B. The overall response rate in this study was 70%, with 11 of 16 assessable patients achieving either a complete (three patients) or partial (eight patients) remission. CONCLUSION: This combination of GM-CSF and PBPC infusion represents an effective method for delivering multiple cycles of high-dose carboplatin chemotherapy and may serve as a model for the administration of high-dose chemotherapy in future trials.     

Objective criteria for in-vitro responses in human tumor colony-forming assays

The application of rigorous statistical criteria to the design and analysis of in-vitro assay techniques can lead to more reliable assessments of their utility. The authors demonstrate that an objective criterion for in-vitro drug sensitivity would yield less pronounced false-negative error rates than a fixed-cutoff rule, and would therefore be more suitable for screening of putative chemotherapeutic agents.     

Gamma-interferon modulates human monocyte/macrophage transferrin receptor expression

Although circulating human monocytes do not express transferrin (Tf) receptors, cultured adherent blood cells display high-affinity Tf binding sites. In the present studies, effects of various cytokines and biologically active proteins on human monocyte/macrophage Tf receptors were investigated. After culture, Tf receptor expression by adherent blood cells was time dependent and plateaued by 7 days. The addition of interleukin-1 (IL-1), alpha-interferon (alpha-IFN), granulocyte/macrophage-colony stimulating factor (GM-CSF), or human IgG to macrophages cultured for 4 days did not alter Tf receptor expression. Fe-saturated, human Tf caused a significant, dose-dependent decrease in receptor expression. At a dose of 100 U/mL, gamma- interferon (gamma-IFN) significantly increased Tf receptor expression by macrophages cultured for 4 (230% +/- 51% of control) or 7 days (150% +/- 22%). Scatchard analyses showed increased binding sites but no change in receptor affinity. Northern and slot blot analysis of cellular mRNA from macrophages cultured for 4 to 7 days and exposed to gamma-IFN showed a two- to fivefold increase in Tf receptor mRNA, but less than or equal to 30% increase in beta-actin mRNA. Ferritin content of gamma-IFN-treated macrophages was 47% to 63% of control cells. Net uptake of 59Fe from Tf by gamma-IFN-treated cells was 10% to 17% of control, but uptake of radiolabeled Tf was comparable. When macrophages were labeled with 59Fe and then exposed to gamma-IFN, cell-associated Fe was reduced by 43%, indicating that gamma-IFN caused macrophage Fe release. gamma-IFN specifically modulates Tf receptor display by inducing Fe release and reducing cellular Fe content. Regulation of Tf receptor expression in macrophages is controlled by cellular Fe content and is thus similar to regulatory mechanisms in dividing cells.     

Effect of interferon on colony formation in culture by blast cell progenitors in acute myeloblastic leukemia

The effect of purified human fibroblast interferon on primary and secondary colony formation by blast progenitors from the peripheral blood of patients with acute myelogenous leukemia was examined. Interferon inhibited blast progenitors and normal granulocyte/macrophage progenitors (CFU-C) in a dose-dependent manner. The magnitude of this effect on blast progenitors and CFU was similar. Interferon also inhibited secondary plating of blast progenitors (self- renewal). This effect was in marked contrast to the effect of adriamycin, which reduced primary plating efficiency of blast progenitors but did not affect self-renewal. Inhibition of blast progenitor proliferation by interferon was markedly reduced when interferon was added after 24 hr of culture and was absent when added after 72 hr. Inhibition of self-renewal was observed even when interferon was added at 72 hr. We conclude that interferon inhibits both primary proliferation and self-renewal of blast progenitors and that this effect is not due to reduction in the number of primary colonies. These experiments provide an example of how cell culture techniques may be used to test antitumor agents for effects on important cellular events other than general cytotoxicity.