Sensitivity of leukemia cell lines to cytotoxic alkyl-lysophospholipids in relation to O-alkyl cleavage enzyme activities.

The human leukemia cell lines K562, HL60, and Raji and the mouse leukemia cell line L1210 showed a differential susceptibility to the action of the alkyl-lysophospholipid (ALP) 1-octadecyl-2-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3). After 48 hours, the 50% growth-inhibition doses (ID50) of ET-18-OCH3 were found to be 0.78 microgram/ml (HL60), 1.53 microgram/ml (Raji), 4.41 micrograms/ml (K562), and 5.05 micrograms/ml (L1210), as determined by [3H]thymidine incorporation. At the same time, cell viability was determined by trypan blue exclusion and revealed median lethal doses (LD50) of 3.5 micrograms/ml (HL60), 15 micrograms/ml (Raji), 24 micrograms/ml (L1210), and 38 micrograms/ml (K562). Since O-alkyl cleavage enzyme previously was suggested as being important in the detoxification of cytotoxic ALPs, the enzyme activity was compared with the susceptibility to ET-18-OCH3 in the distinct cell lines. In comparison to an approximate sevenfold to elevenfold (ID50 and LD50, respectively) difference in the susceptibility of the above leukemia cell lines to ET-18-OCH3, no significant difference in the specific activities (0.13-0.21 nmol/min/mg) of the O-alkyl cleavage enzyme was found in the above leukemia cell lines. Therefore, the differential sensitivity of the above lines investigated cannot be explained by differences in O-alkyl cleavage enzyme activity. Experiments with radiolabeled ET-18-OCH3 in Raji cells suggest, rather, a critical role for phospholipases C and/or D in ALP metabolism.

Phase II study of aminoglutethimide and medroxyprogesterone acetate in the treatment of patients with advanced breast cancer.

Forty-five women with far-advanced metastatic breast cancer were treated with a combination of aminoglutethimide (AG), 1000 mg p.o. daily, and medroxyprogesterone acetate (MPA), 1500 mg p.o. daily. Of 41 patients evaluable for treatment response, there were two complete responses, five partial remissions, 26 patients with minor tumor responses or no change, and eight nonresponders. Major side effects included those known for AG and MPA, i.e., impairment of mental functions, depressive syndromes, fatigue, ataxia, skin rash, changes in body weight, and transient increase of gamma-glutamyl-transferase. Most side effects disappeared spontaneously after 4 to 6 weeks of treatment. Plasma hormone measurements in 28 patients revealed no impairment of adrenocorticotropic hormone and cortisol levels. In conclusion, in the AG combination, it is feasible and safe to replace cortisol by MPA. Treatment results warrant further investigation of AG-MPA in patients with breast cancer of a more favorable prognosis.

Pattern of endogenous lectins in a human epithelial tumor.

Salt and detergent extracts of a malignant epithelial tumor, obtained by extraction of acetone powder, were fractionated on different sets of Sepharose columns covalently derivatized with lactose, asialofetuin, melibiose, mannan, fucose, and heparin. Successive elution by chelating reagent and specific sugar resulted in isolation of different Ca2+-dependent and Ca2+-independent endogenous carbohydrate-binding proteins, as analyzed by gel electrophoresis. It appears from the analysis that certain bands represent newly identified proteins capable of binding to lactose (at Mr 64,000), melibiose (at Mr 28,000), and fucose (at Mr 62,000 and 70,000). Other carbohydrate-binding proteins isolated from this human tumor have been identified in normal, especially embryonic, tissues of different nonhuman vertebrates. The carbohydrate-binding proteins are assayable as agglutinin with rabbit erythrocytes and show no detectable enzymatic activity. They can thus be defined as lectins. The presence of a complex pattern of endogenous lectins and their biochemical characteristics may contribute to an understanding of intercellular interaction during the complex process of metastatic spread and may furthermore allow a new tool for diagnosis and a lectin-based therapy.

New assay for the O-alkyl cleavage enzyme with alkyl-lysophospholipids as substrates.

The O-alkyl cleavage enzyme is important for the metabolism of cytotoxic alkyl-lysophospholipids. We have developed a simple new method for the determination of the enzyme activity which is based on the formation of water-soluble phosphate during the enzyme reaction. This is the first assay which avoids the use of radiolabeled substrates.

Biological and biochemical characterization of a somatostatin-producing human carcinoma established by heterotransplantation.

A somatostatin-producing human carcinoma cell line was established by heterotransplantation into athymic nude mice. The original material, which was derived from a colon tumor of a patient who had previously had bilateral ovarian tumors contained 66 ng extractable somatostatin/g tissue. Somatostatin-producing cells could be identified by immunohistochemistry within the first tumor transplants. Although initially the somatostatin concentration was low (14 ng/g) a progressive increase was observed with each successive transplantation so that after 10 passages it reached a level of 127 ng/g tissue. Analysis of tumor extracts by gel filtration and high-performance liquid chromatography indicated that somatostatin-14 was the only molecular form produced by the original and by the transplanted tumor after multiple passages. This result demonstrates that the tumor has the ability to constitutively express the prosomatostatin gene and to process the primary translation product to somatostatin-14.

Differential gamma-interferon response of human colon carcinoma cells: inhibition of proliferation and modulation of immunogenicity as independent effects of gamma-interferon on tumor cell growth.

The effect of recombinant gamma-interferon (IFN-gamma) on established human colon carcinoma cell lines as well as fresh tumor cells from colon carcinoma patients has been investigated with respect to growth inhibition, enhancement of HLA expression, and modulation of immunogenicity. A direct antiproliferative activity of IFN-gamma was observed in five of seven cell lines tested, with a reduction of [3H]thymidine incorporation between 30 and 90%. Depending on the cell line, the IFN-gamma doses required for maximal inhibition varied between 20 and 2 X 10(4) units/ml. Independent of this effect, IFN-gamma enhanced the expression of HLA-A,B,C antigens in all cells investigated and induced expression of HLA-DR in three of seven carcinoma cell lines. Antigenic modulation of Class I and II major histocompatibility complex antigens was paralleled by an enhancement of the in vitro immunogenicity in three of four established carcinoma lines and in three of three cases, using cells derived from primary tumor cultures. Induction or enhancement of both proliferative and cytolytic T-cell responses was obtained in allogeneic and in autologous mixed-lymphocyte tumor cell cultures.

Steady-state and dynamic properties of cardiac sodium-calcium exchange. Sodium-dependent inactivation.

Sodium-calcium exchange current was isolated in inside-out patches excised from guinea pig ventricular cells using the giant patch method. The outward exchange current decayed exponentially upon activation by cytoplasmic sodium (sodium-dependent inactivation). The kinetics and mechanism of the inactivation were studied. (a) The rate of inactivation and the peak current amplitude were both strongly temperature dependent (Q10 = 2.2). (b) An increase in cytoplasmic pH from 6.8 to 7.8 attenuated the current decay and shifted the apparent dissociation constant (Kd) of cytoplasmic calcium for secondary activation of the exchange current from 9.6 microM to < 0.3 microM. (c) The amplitude of exchange current decreased synchronously over the membrane potential range from -120 to 60 mV during the inactivation, indicating that voltage dependence of the exchanger did not change during the inactivation process. The voltage dependence of exchange current also did not change during secondary modulation by cytoplasmic calcium and activation by chymotrypsin. (d) In the presence of 150 mM extracellular sodium and 2 mM extracellular calcium, outward exchange current decayed similarly upon application of cytoplasmic sodium. Upon removal of cytoplasmic sodium in the presence of 2-5 microM cytoplasmic free calcium, the inward exchange current developed in two phases, a fast phase within the time course of solution changes, and a slow phase (tau approximately 4 s) indicative of recovery from sodium-dependent inactivation. (e) Under zero-trans conditions, the inward current was fully activated within solution switch times upon application of cytoplasmic calcium and did not decay. (f) The slow recovery phase of inward current upon removal of cytoplasmic sodium was also present under the zero-trans condition. (g) Sodium-dependent inactivation shows little or no dependence on membrane potential in guinea pig myocyte sarcolemma. (h) Sodium-dependent inactivation of outward current is attenuated in rate and extent as extracellular calcium is decreased. (i) Kinetics of the sodium-dependent inactivation and its dependence on major experimental variables are well described by a simple two-state inactivation model assuming one fully active and one fully inactive exchanger state, whereby the transition to the inactive state takes place from a fully sodium-loaded exchanger conformation with cytoplasmic orientation of binding sites (E1.3Ni).

Autologous marrow recovery following allogeneic marrow transplantation in a patient with severe aplastic anemia.

A 45-year-old woman with severe idiopathic marrow failure was prepared for marrow transplantation by administration of cyclophosphamide (cy) 50 mg/kg on each of four successive days. She then received an intravenous infusion of 20 X 10(9) nucleated marrow cells from an HL-A matched and mixed lymphocyte culture (MLC) non-reactive sister. There was evidence for minimal marrow recovery in 1-2 months and a second marrow infusion was carried out 69 days after the first without additional immunosuppression. There was a continued slow recovery of peripheral blood counts with complete reconstitution of erythropoiesis, return of the white blood cell count to between 3 and 4000/mm3, with 50% granulocytes, and platelets to 60--70,000/mm3, 11 months after the initial grafting attempt. Red cell antigens and gamma globulin allotypes were of recipient type. The MLC and the indirect cell mediated lympholysis (CML) test became positive possibly indicating cellular sensitization to non HL-A antigens. This report of a patient with severe marrow failure documents autologous recovery of marrow function after receiving a large dose of cy and allogeneic marrow. The implications of this are discussed.

Resistance of tumour cells to chemotherapy: importance of host defence factors.

The arguments favouring the hypothesis that chemotherapeutic agents might act in cooperation with host defence mechanisms are reviewed briefly. In patients with far advanced solid tumours plasma factors blocking in vitro immune reactions have been identified and successfully removed by immune adsorption or plasma exchange. By plasmapheresis performed in patients with metastatic malignancies resistant to chemotherapy it was possible to induce tumour regressions. In 25/28 patients responding to the combined plasmapheresis/chemotherapy procedure a positive correlation was found to clinical results and patterns of plasma-blocking factor activities.

Double-blind randomised trial of the antiemetic efficacy and safety of ondansetron and metoclopramide in advanced breast cancer patients treated with epirubicin and cyclophosphamide.

Ondansetron was compared with metoclopramide for antiemetic efficacy in a randomised double-blind trial in 122 patients with advanced breast cancer. All patients were treated with epirubicin (greater than 50 mg/m2) and cyclophosphamide (greater than 500 mg/m2). 50 patients receiving ondansetron and 60 with metoclopramide were considered evaluable. Ondansetron was at least as effective as metoclopramide in the control of vomiting and nausea. The percentage of patients with complete plus major control was 72% (59-85%) vs. 61% (48-74%) on day 1 (P = 0.230) and 79% (67-91%) vs. 66% (53-78%) on days 2-3 after chemotherapy (P = 0.122). Over the 3-day study period, nausea was absent or mild in 60% of the patients treated with ondansetron, compared to 45% given metoclopramide (P = 0.064). No major drug-related side-effects were reported. 1 patient receiving ondansetron experienced gastrointestinal disturbance and headache. Episodes of diarrhoea, fever, hyperkinetic syndrome, fatigue, restlessness and migraine with vomiting were reported by 5 patients treated with metoclopramide. None of the changes in the biochemical or haematological parameters was attributed to the antiemetic treatments.

Chromatofocusing combined with the ELISA technique. A sensitive method for the analysis of immune complexes.

A sensitive method which permits analysis of IgG containing circulating immune complexes without detailed knowledge of the nature of the antigens and the specificity of the antibodies involved is described. Soluble BSA: anti-BSA were used as model immune complexes and isolated from serum. The procedure involves the use of gel chromatography for the separation of the high molecular weight fraction containing the immune complexes as measured by binding to 125I-labeled Clq, followed by absorption of the immune complex fraction to immobilized protein A-Sepharose CL-4B. After desorption from protein A-Sepharose the complexes were dissociated and separated into free antigen and antibody by chromatofocusing in the presence of urea. The isolated free antigen and antibody retained their immunological activity as shown by immunodiffusion, binding after their recombination to 125I-labeled Clq, and by recombining antigen and antibody with much enhanced sensitivity using a microplate ELISA system. By means of the ELISA recombination technique it is possible to analyze less than 1 microgram of BSA:anti-BSA model complexes. Application of this technique may provide more information about the nature of immune complex like material associated with diseases.

Metastatic breast cancer with constantly low CEA blood levels. A subgroup with unfavorable prognosis?

The capability of breast cancer to secrete CEA might have biological significance. In 105 patients with metastatic breast cancer serial CEA determinations and clinical follow-up data were available during progression of disease up to death. In this series, 39 patients (37%) had constantly low CEA levels (less than 10 ng/ml), whereas 66 patients (63%) showed CEA values exceeding 10 ng/ml with progression. The patients with low CEA levels had significantly shorter median survival times (P = 0.001) after mastectomy (39 versus 65 months) and after recurrence (18 versus 28 months) than the patients with high CEA levels. This difference was due first to a poor-risk group of 13 patients with rapidly disseminating tumors, very short survival (less than 12 months), and low CEA levels. Secondly, there were more patients with pulmonary involvement and unfavorable prognosis and fewer patients with osseous metastases and long survival in the low-CEA group. In conclusion, there might be a subtype of breast cancer with rapid progression and low CEA secretion. This clinical observation has to be confirmed by histological grading and CEA staining of these tumors.

Effect of ether lipids on mouse granulocyte-macrophage progenitor cells.

In this study we determined the potential bone marrow toxicity of the ether lipid derivatives 1-0-octadecyl-2-0-methyl-rac-glycero-3-phosphocholine (OcMe-G-3-PC), 1-0-hexadecyl-propanediol-2-phosphocholine (He-Pr-2-PC), and hexadecylphosphocholine (He-PC). OcMe-G-3-PC inhibited the proliferation of mouse granulocyte-macrophage progenitor cells (GM-CFCs) at a dose of 1 micrograms/ml, whereas He-Pr-2-PC and He-PC started to inhibit the growth of hemopoietic precursors at 5 micrograms/ml. In contrast to this finding, NMRI mice given 10 mg/kg i.v. daily for 4 weeks and 20 or 30 mg/kg for 5 days showed no bone marrow toxicity. We conclude that the dose-dependent toxic effects observed in vitro are within the physiological tolerance in vivo.

Vindesine. A clinical trial with special reference to neurological side effects.

A good tumoricidal activity of vindesine (VDS) has been reported in a variety of animal tumors and in human leukemias and lymphomas. We treated 22 patients who had received no prior chemotherapy and were suffering from a variety of malignant neoplasms with 0.5 mg/m2 to 3.0 mg/m2 VDS i. v. once or three times at weekly intervals and recorded the clinical, hematologic, and especially, neurological side effects. Clinically we observed fatigue in nine patients, paresthesias in seven, myalgias in three, vertigo and diarrhea in two, and skin pains, tinnitus, gastric pains, alopecia, and tremor in one patient each. There was no obvious dose-action relationship. Paravenous injection caused cellulitis similar to that seen with vincristine. No side effects were apparent in liver (SGPT) and renal (creatinine) function tests. Hematologically there was a clear trend toward leukopenia with higher doses of DVA and a mean increase in the thrombocyte count by 51 X 10(3)/mm3 was found (sign test: P greater than 0.05). The hemoglobin level did not change. Clinical neurological examination and monitoring by electroneurography revealed no changes in tensiometer performance, motor and sensory nerve conduction velocity, motor or sensory nerve action potential amplitudes, or H-reflex responses. There was dose-related diminution of the proprioceptive reflexes, especially in the lower extremities. Even with as little as 2.0 mg/m2 VDS i. v. at weekly intervals for 3 weeks Achilles and patellar tendon reflexes were diminished or absent in all patients.

Phase II study with etoposide in previously untreated advanced breast cancer.

A phase II study was carried out to evaluate the efficacy and safety of etoposide used as first-line chemotherapy for patients with advanced breast carcinoma. A total of 20 patients received 230 mg/m2 i.v. etoposide per day for 3 days (total, 690 mg/m2 per course) every 4 weeks. A total of 95 courses were given. Observed responses included 3 partial remissions (PR) and 14 cases of stable disease (NC). The median duration of response was 6 (PR) and 5.6 months (NC). Contrary to the severe hematological toxicity in heavily pretreated patients described in previous studies, no substantial problems were observed in this trial. No dose reduction was necessary, and only once did leukopenia lead to a 1-week delay in therapy. An increase in platelets up to a maximum of 685,000/mm3 was seen in all patients, particularly in those with bone metastases. No relation to the quality of remission or pretreatment was seen. Nausea, vomiting, and fatique were mild and transient, but alopecia occurred in all cases. One patient developed nonfatal anaphylactic shock after etoposide treatment.

Intra-arterial perfusion therapy with 5-fluorouracil in patients with metastatic colorectal carcinoma and intractable pelvic pain.

In a phase-II trial, 18 patients with intractable pelvic and perineal pain caused by local recurrent and/or metastatic colorectal carcinoma resistant to combinations of analgesics, systemic cytostatic chemotherapy and/or radiation were treated with intra-arterial perfusion therapy using 15-30 mg 5-FU/kg body wt./day for 1-5 days. Of 18 patients, ten achieved complete pain relief for 3-32 weeks (mean, 15.7 weeks); after the perfusion therapy eight used less than 50% of the amount of analgesics required before treatment; one patient had only a minor response; two patients were treated unsuccessfully. Side effects were mild and controllable. One patient died subsequent to arterial embolism in the leg where the catheter was placed; pelvic perfusion therefore appears risky in patients with severe arteriosclerosis.

Evidence for the involvement of protein-carbohydrate interaction in hematopoietic, multipotential colony-stimulating factor-dependent stem cell proliferation.

Immunologically important mediators have been shown to exhibit ability to specifically bind distinct carbohydrates. This type of protein-carbohydrate interaction is one mechanism how to explain involvement of glycochemical interactions in regulatory processes. Interference of certain saccharides with murine multipotential colony-stimulating factor (multi-CSF)-dependent colony formation from progenitor cells in semisolid agar raised evidence for similar potential involvement of protein-carbohydrate interactions. Affinity depletion of conditioned WEHI-3B-medium on resins, bearing saccharides that have been elucidated to be effective inhibitors (mannose and lactose), resulted in preparations with significantly reduced capability to sustain development and proliferation. Sequence comparison of multi-CSF to carbohydrate-binding proteins (lectins) with this specificity failed to uncover extended homologies in diagonal plots. But detailed sequence alignments revealed confined, high-scoring stretches of homology between various lectins and two types of CSF. These results prove the importance of protein-carbohydrate interactions in stem cell proliferation.

Mastectomy stimulates prolactin release in breast cancer patients.

Hyperprolactinaemia has been described to occur after mastectomy in breast cancer patients, but whether it may be the result of surgery or breast cancer is particularly unknown. Plasma prolactin levels were measured in 51 patients one day before, and 1, 7, 30, and 180 days after mastectomy (23 primary breast cancer patients), tumourectomy (10 patients with benign or malignant breast lesions), and cholecystectomy (18 patients with cholelithiasis). Elevated prolactin levels were found on the 7th and 30th postoperative day in mastectomized and laparotomized patients, but not in patients who underwent tumourectomy of benign or malignant breast lesions (p less than 0.01). The prolactin levels were in the normal range one day before and again 180 days after surgery in all patients. We conclude, therefore, that postoperative hyperprolactinaemia in breast cancer patients is a result of surgery rather than the disease.

Characterization of membrane lectins in human colon carcinoma cells by flow cytofluorometry, drug targeting and affinity chromatography.

Flow cytofluorometry and drug targeting with labelled neoglycoproteins are used as tools to probe for membrane lectins in two human adenocarcinoma cell lines. Both cell lines express activities for galactosides, glucosides and fucosides. Affinity chromatography on gels with immobilized sugar leads to purification of an alpha-galactoside-binding protein at an apparent molecular weight of 64 kDa that also binds to lactose, maltose and fucose and exhibits Ca2+-requirement for binding, a beta-galactoside-binding protein without Ca2+-requirement at an apparent molecular weight of 14 kDa, and an alpha-glucosyl-binding protein without Ca2+-requirement at an apparent molecular weight of 34 kDa from both cell lines. The description of membrane lectins, documented here for the first human tumor cell lines, is an initial step towards a lectin-based improvement of the clinical management of human colon adenocarcinoma.

Xenografts from a human colon carcinoma and its metastases: establishment, characterization and differences in the pattern of carbohydrate-binding proteins.

Investigation of the pathogenesis of human colorectal carcinoma metastasis can be rendered experimentally possible by suitable human cell biological model systems. The purpose of these studies was to establish xenografts in nude mice from human colon carcinoma and from its metastasis in the same patient as an appropriate model. Surgically removed biopsy specimens from a colon adenocarcinoma (grade 3) and its local relapse two years later with metastases in the small intestine were established as xenotransplants and their growth characteristics examined. Both tissue types shared common characteristics with respect to marker expression (carcinoembryonic antigen, neuron-specific enolase, cytokeratin). The primary tumor showed remarkable development of necrotic effusion with cytotoxic activity that ceased after several passages. The profile of endogenous carbohydrate-binding proteins (lectins), the receptors for cellular glycoconjugates in a recognitive protein-carbohydrate interplay with potential relevance to metastases formation, revealed differences between these two human tumor samples of identical origin, especially with respect to beta-galactoside-specific receptors. This glycobiochemical analysis employed standardized procedures. Prolonged passaging was also shown to result in profile alterations, as was similarly noted in comparison to another species. These studies may encourage the application of systems of primary tumor and its metastases in the same patient in attempts to correlate the expression of cellular characteristics with the biological and clinical behavior of human colonic tumor cells.