Takao Ohnuma

Inhibitory Effects of Human Leukocyte and Fibroblast Interferons on Normal and Chronic Myelogenous Leukemic Granulocytic Progenitor Cells

Inhibitory effects of two human interferon preparations, leukocyte interferon (Le-IF) and fibroblast interferon (F-IF), on granulocytic progenitor cells (CFU-C) from hematologically normal cancer patients and from patients with chronic myelogenous leukemia were evaluated. There was a wide variation in sensitivity of CFU-C to both Le-IF and F-IF. F-IF was mor inhibitory against CFU-C than le-If. Normal CFU-C and chronic myelogenous leukemia CFU-C were equally inhibited by both interferons. Effects of both interferons were neutralized by corresponding specific antisera but not by the other antisera. These observations confirmed that differences in immunogenicity of the interferons may attend their different origins.

Marker Profiles of Human Leukemia and Lymphoma Cell Lines

SummaryBy means of the multiple marker analysis, a total of 55 human leukemia-lymphoma cell lines which included 15 T-cell, 30 B-cell, four myelomonocytic-cell, and six non-T, non-B cell lines was characterized for their marker profiles. The multiple markers used included a number of cell surface markers as detected by either rosette or immunofluorescence tests, enzyme assays, cytogenetic analysis, and certain functional assay. Based on the criteria previously defined it was found that all the cell lines were proved to represent original leukemia-lymphoma of ALL, AML, CLL, CML in blastic phase or variety of lymphomas. The monoclonality, a “frozen” state at a specific stage of differentiation-maturation, and cytogenetic marker in each leukemia-lymphoma cell line were remarkable common properties and were stable for years of cultivation. Similar, if not identical, general characteristics were observed in the study on 344 cases of uncultured fresh leukemia-lymphomas by the multiple marker analysis. While no single marker specific to any type of tumor was found, the study offers not only a basis for better understanding of the biology of leukemia-lymphoma but also an insight into normal hematopoietic cell differentiation in man.

Effects of hyaluronidase on doxorubicin penetration into squamous carcinoma multicellular tumor spheroids and its cell lethality

Doxorubicin is an anticancer agent widely used in the treatment of human cancer. The major limitation of this drug governing the cell-killing effect appears to be its poor penetration into a tumor mass. We have studied the effects of hyaluronidase on the penetration and cell-killing effect of doxorubicin using multicellular tumor spheroids (MTS). MTS approximately 500 μm in diameter were produced by a liquid-overlay culture technique from PC-10 lung and HEp-2 laryngeal squamous carcinoma cell lines. Cells in MTS and monolayer were exposed to hyaluronidase for various lengths of time; this was followed by a 1-h resting interval and a subsequent 1-h exposure to doxorubicin. MTS and monolayer cells were then trypsinized to a single-cell suspension and subjected to clonogenic assay. Hyaluronidase at a concentration of 25 U/ml or 250 U/ml was nontoxic to the monolayer cells. For PC-10 MTS, pretreatment with 25 U/ml hyaluronidase for 24 h and 72 h resulted in approximately 20% increases in Doxorubicin cell killing at the median (IC50) dose as compared to doxorubicin alone. HEp-2 MTS were more sensitive to the hyaluronidase pretreatment. Thus, a 1-h exposure to the enzyme produced a 40% increase in doxorubicin-induced cell death at the IC50 dose. A fluorescence microscopic study revealed that a 1-h exposure of MTS to doxorubicin produced doxorubicin fluorescence only in the one or two outer layers of MTS. When MTS were pretreated with hyaluronidase, there was enhanced penetration of doxorubicin fluorescence into the MTS core. Hyaluronidase-induced enhancement of Doxorubicin penetration and its cell-killing effect is dependent on the exposure time and tumor cell origin. These data suggest that anecdotal reports of hyaluronidase-enhanced activity of preclinical chemotherapy deserve a controlled trial.

Phase 1 study of carboplatin in patients with advanced cancer, intermittent intravenous bolus, and 24-hour infusion.

We undertook a phase 1 study of Carboplatin (CBDCA) on an intermittent single intravenous (IV) bolus (schedule A) and a 24-hour continuous infusion schedule (schedule B). Hydration and forced diuresis were not performed. Patients were not premedicated for anticipated vomiting. Thirty-eight adult patients with solid tumors received a total of 71 courses. In schedule A, doses were escalated from 20 to 600 mg/m2. The dose-limiting toxicity was myelosuppression. At doses of 270 mg/m2 and higher, leukopenia and thrombocytopenia were reproducibly seen. The dose of 600 mg/m2 was the maximally tolerated dose, producing severe thrombocytopenia (platelet counts less than 30,000/microL). Other toxicities included a fall in hemoglobin levels and tolerable nausea and vomiting. Schedule B produced comparable hematologic and emetogenic toxicities to those in schedule A. In three patients audiograms became abnormal with high-frequency hearing loss without overt deafness. Two patients developed hypomagnesemia without irreversible renal dysfunction. Patients with poor performance status, preexisting renal dysfunction, a third fluid space, or bone metastases seemed to develop increased hematologic toxicity. The recommended phase 2 dose for good risk patients is 400 mg/m2 IV bolus and for poor risk patients 270 mg/m2 IV bolus. Responses were seen in one patient each with head and neck carcinoma (partial response), small cell lung cancer (minor response), and breast cancer (minor response).

Erwinia carotovora asparaginase in patients with prior anaphylaxis to asparaginase from E. coli.

Asparaginase prepared from Erwinia carotovora was well tolerated when given to four patients with acute lymphocytic leukemia who previously had had anaphylactic reactions from E. coli asparaginase. The Erwinia enzyme was cleared from plasma with a half life of 7 to 13 hours, a time similar to some preparations of E. coli asparaginase in patients who had not sustained anaphylaxis. Serum asparagine levels decreased to unmeasurable levels after Erwinia asparaginase in a manner reminiscent of the E. coli enzyme. No positive precipitin reaction or skin test to either E. coli or Erwinia enzyme was detected. These results indicate that there is no great degree of immunologic cross reaction to prevent the use of the Erwinia enzyme in patients who sustained anaphylaxis to the E. coli preparation. The availability of a second active asparaginase from a different microbiological source extends the usefulness of asparaginase in clinical therapy. Stabilization and protective effect of Erwinia enzyme activity by human plasma was demonstrated.

Diabetes in patients treated with asparaginase.

In 5 patients treated with L‐asparaginase, plasma insulin response to intravenous glucose injection was measured by radioimmunoassay. Two of the patients developed a diabetic state (2 and 4 days) after a single dose of L‐asparaginase, while a third patient developed diabetes 2 days after the fourth dose of L‐asparaginase. Plasma insulin was not detectable by radioimmunoassay during the diabetic state in each of them. The normal pattern of insulin response returned completely in one of the 3 patients within 23 days, whereas suboptimal response persisted for 2 weeks and 9 months in the remaining 2 patients. The two remaining patients were studied prior to and 6 days after a single dose of L‐asparaginase; neither developed a diabetic state.

Clinical study with bleomycin: Tolerance to twice weekly dosage

Fifty‐nine patients with inoperable malignant neoplasms were treated with bleomycin at dose levels ranging rom 1.25 mg/m2 to 35 mg/m2 of estimated body surface area twice weekly in an attempt to define host tolerance to this new antineoplastic agent. The drug was given for a total of 12 doses, if tolerated, and longer in the presence of objective tumor response. At 4 mg/m2, chills and fever, vomiting, and moderate cutaneous toxicity appeared. At 15 mg/m2, 3 of 11 patients developed dose‐limiting skin toxicity. Two additional patients developed pulmonary infiltrates after prolonged courses of treatment to a total of 546 and 582 mg, respectively. At 26 mg/m2 and higher, prohibitive pulmonary and hematologic toxicity developed during the initial course of study requiring premature termination of scheduled courses. At 35 mg/m2, transient hypertension, confusion, abdominal distention, and urinary burning also developed. Four of 19 patients with squamous cell carcinoma of the head and neck, one patient with squamous cell carcinoma of the skin, 3 of 6 patients with lymphosarcoma, and 1 of 4 patients with testicular carcinoma responded with more than 50% tumor regression. One patient with lymphosarcoma was induced into complete remission. Minimal therapeutic dose levels for lymphosarcoma and for squamous cell carcinoma were 4 mg/m2 and 6 mg/m2, respectively. This study indicates that optimal dose levels range between 4 and 15 mg/m2 twice weekly. The present study establishes patient tolerance to twice weekly doses of bleomycin and confirms therapeutic benefit for squamous cell carcinoma and lymphosarcoma over a remarkably wide range of doses.

Biologic and pharmacologic effects of harringtonine on human leukemia-lymphoma cells

SummaryTen human leukemia-lymphoma cell lines were tested for the growth-inhibitory effects of harringtonine (HT). HT was most active against HL-60 acute promyelocytic leukemia cells and least active against DND-41 acute lymphoblastic leukemia cells, with a 70-fold differential activity. Sensitivity of the cell lines is, in decreasing order: HL-60 > RPMI-8402 > DND-39A ≃ ML-2 ≃ MOLT-3 ≃ KG-1 > Daudi ≃ NALL-1 > BALM-2 > DND-41. The cell lines with rapid cell growth tended to be more sensitive to HT. To further elucidate the selectivity of the differential sensitivity, uptake and release of HT were compared in HL-60 and DND-41 cells. Uptake of [3H]HT into HL-60 and DND-41 cells showed no difference; however, the binding of [3H]HT to cellular components was > 16-fold higher in HL-60 cells than DND-41 cells. There were also minor, but significant differences in the inhibition of [3H]leucine incorporation into proteins of these two cell lines in the presence of 1 μg/ml HT. To test whether the biological effects of HT are related to the concentration of, or exposure time to, HT, KG-1 cells were exposed to HT for different periods of time and the growth-inhibitory effects were compared. Increasing exposure time from 1 h to 3 h resulted in a 100-fold decrease in concentration x exposure time (c x t) at ID50; from 3 h to 6 h, in a 20-fold decrease at ID70; and from 6 h to 24 h, in a 16-fold decrease at ID90. HT was not inactivated by cells up to 24 h. These results indicate that (a) the sensitivity of different cell lines to HT may be related to the degree of HT binding; and (b) the effects of HT are more dependent on exposure time than concentration. Continuous infusion is thus rational for clinical trials of this drug, and the degree of HT binding to leukemic cells may be predictive of clinical response.

Retrovirus-Mediated Transfer of Anti- MDR1 Ribozymes Fully Restores Chemosensitivity of P-Glycoprotein-Expressing Human Lymphoma Cells

Development of multidrug resistance (MDR) is the major obstacle to successful cancer chemotherapy. We have developed Daudi human lymphoma cells that are 20-fold more resistant than the parent cell line to vincristine (VCR) by infecting cells with pHaMDR1/A retroviral vector (Daudi/MDR20). Three DNA sequences of anti-MDR1 hammerhead ribozymes (Rzs), one cleaving codon 196 of MDR1 mRNA (196MDR1-Rz), the second a stem II base-modified (U9-->Gg, U13-->A13, G14-->A14, A18-->C18) Rz against codon 196 (196MDR1-sRz), and the third a stem II base-modified Rz directed against the -6 approximately -4 GUC sequence of the translation initiation site of the MDR1 mRNA (iMDR1-sRz), were synthesized and cloned into the retroviral vector N2A+tRNAiMet downstream of the RNA polymerase III promoter and adjacent to a tRNA gene sequence, forming the constructs N2A+tRNAiMet-196MDR1-Rz, N2A+tRNAiMet-196MDR1-sRz, and N2A+tRNAiMet-iMDR1-sRz. The three constructs were transfected into GP+envAM 12 cells for packaging the retroviral vectors. The supernatants containing the packaged retrovirus in high titers (1.1-2.5 X 10(5) CFU/ml as determined by infection of NIH 3T3 cells) were used to infect Daudi/MDR20 cells. The iMDR1-sRz- and 196MDR1-sRz-transduced Daudi/MDR20 cells completely restored chemosensitivity to VCR and doxorubicin, and were accompanied by blocked expression of MDR1 mRNA and P-glycoprotein as well as overexpression of anti-MDR1 Rz. In a cell-free system, the chimeric tRNA-sRz molecules were more stable and had more efficient catalytic activities than the corresponding naked Rz molecules. The stem II base-modified Rz were also more stable and efficient in catalytic activities than the unmodified Rz molecules. The base modification in the Rz stem II structure and the development of chimeric tRNA-Rz molecules were identified to enhance the cleavage efficacy. The combination of these two factors, together with the use of a retroviral vector, appear to have contributed to the complete reversal of MDR.

Doxorubicin plus cisplatin in the treatment of apudomas.

Twelve patients with advanced apudomas—six with carcinoid tumors, two with chemodectomas, two with pancreatic islet cell tumor, and one each of medullary carcinoma of the thyroid and paraganglioma of unknown primary—were treated with a combination of doxorubicin 50 mg/m2 and cisplatin 50 mg/m2 every 3 to 4 weeks. Biochemical markers were present in 8 of the 12 patients. Five of the 12 patients (3 with carcinoid and 2 with chemodectomas) responded with more than 50% regression of tumor size measured as hypothetical area. Three others (two with islet cell tumors and one with carcinoid) had clinical and/or biochemical improvements. A median duration of response was 6 months. Nausea, vomiting, and alopecia were universal. Mild or moderate leukopenia was the most frequent toxicity. No sustained nephrotoxicity was seen. The combination of doxorubicin and cisplatin provides a new palliative therapy for patients with APUD tumors.