Bromodeoxyuridine in tumors and chromosomes detected with a monoclonal antibody.

Using a monoclonal antibody to bromodeoxyuridine (BUdR) and immunohistochemistry, we measured the incorporation of this thymidine analogue into the DNA of human normal and malignant cells exposed in vivo. BUdR given as a constant intravenous infusion for 12 or 24 h daily for up to 13 d resulted in a steady-state plasma level of 10(-6) M during the infusion. We demonstrated extensive incorporation of BUdR into both normal skin, normal bone marrow, and malignant melanoma cells. In addition, this infusion of BUdR was adequate to identify sister chromatid exchanges from human marrow chromosomes exposed in vivo. Using this constant infusion, significant but reversible (acute) toxicity was observed with myelosuppression and skin photosensitivity. These techniques, which are considerably less cumbersome and time-consuming than the use of radioactive isotopes of thymidine, can be used for further human studies of cell kinetics and chromosomal replication in both normal and malignant cells.

Heterogeneity in the radiation survival curves and biochemical properties of human lung cancer cell lines.

Human lung cancers of distinct histology exhibit different responses to radiation therapy in vivo. For examination of the basis of this phenomenon, the radiation survival curves and levels of relevant enzymes were determined in 16 lung cancer cell lines derived from tumors of different histology. These included lines from 5 adenocarcinomas, 7 small cell tumors, 3 variant small cell tumors, and 1 large cell tumor. These findings were compared to those obtained with the use of a normal skin fibroblast cell line. Whether cloned in liquid culture or soft agarose, cell lines had similar radiation survival curves. These curves were consistent with the apparent in vivo radiation responsiveness of the tumors. Although considerable heterogeneity in radiation survival curves was observed among the cell lines, cells from large cell lines and small variant lines had pronounced shoulders and extrapolation numbers (n) from 5.6 to 14. In contrast, cells from small cell lines and adenocarcinoma cell lines were more "sensitive" (-n values of 1-3.3). In these cell lines, levels of DNA polymerase beta, glutathione (GSH), GSH transferase, GSH reductase (NAD(P)H), gamma-glutamyltransferase did not correlate with radiation parameters of sensitivity. DNA polymerase beta and GSH levels were, however, higher than those in a line of normal skin fibroblasts. These cell lines may be useful in identifying the basis of the variable responsiveness of human lung cancer cells to ionizing radiation.

Hemopoietic growth factors: a review.

Several hemopoietic growth factors have now been purified, cloned, and produced in bacteria. Granulocyte colony stimulating factor and granulocyte-macrophage colony stimulating factor are already being used in clinical trials. Within 12 months two more hemopoietic growth factors, macrophage colony stimulating factor (also called colony stimulating factor 1) and interleukin 3 (also called multi-colony stimulating factor) will be used for patient treatment. This review discusses the recent advances in our knowledge of the molecular properties and biological specificities of these factors. It is now clear that these molecules are able to modulate selectively the activity of mature blood cells as well as stimulating the production of specific lineages of blood cells. The availability of recombinant hemopoietic growth factors purified from animal or yeast cell conditioned medium or bacteria has facilitated in vivo experiments, as well as the clinical trials. Each of the growth factors has a unique spectrum of biological activities and it appears that the growth factors will enhance the recovery and function of circulating white blood cells after cancer therapy or bone marrow transplantation.

Uptake of hematoporphyrin derivative by normal and malignant cells: effect of serum, pH, temperature, and cell size.

Normal and malignant cells were incubated with hematoporphyrin derivative (HPD) and their uptake and retention of HPD were analyzed by flow cytometry. In standard growth medium the amount of HPD taken up by cells was proportional to the added HPD concentration and reached a plateau level after 5-6 h of incubation. The uptake occurred in two steps; within seconds a large amount of HPD became loosely bound to the cells, presumably the outer membrane. This was followed by a slower uptake of HPD into the cytoplasm. The loosely bound portion could be washed from the cells by medium containing either fetal calf serum or serum albumin. At low temperatures the uptake into the cytoplasm was strongly reduced. A major determinant of HPD uptake was the concentration of serum in the medium. At any particular concentration of HPD below 200 mg/liter, increasing concentrations of fetal calf serum or bovine serum albumin resulted in a reduction in the amount of HPD taken up by cells. A further factor affecting uptake was the pH of the medium. At low pH (pH 6) the rate of HPD incorporation was much higher than at pH 7.4. Under identical conditions of incubation, HPD uptake was proportional to cell size as estimated using the low angle light scatter signal in the flow cytometer. Our data suggest that acidic pH, differences in extracellular serum concentrations of malignant tumor tissue, as well as the increased size of tumor cells may play an important role in the selective uptake of HPD by malignant tumors.

Development of a xenograft glioma model in mouse brain.

Xenograft intracerebral glioma models have been developed in normal mice by growing the rat C6 glioma in either adult or neonatal mouse brains. Using this tumor line it was possible to grow discrete intracerebral gliomas in either CBA or AKR adult mice or neonatal mice. The size of the tumor mass and length of survival was directly related to the number of tumor cells injected and the time after implantation. To obtain localized intracranial tumor growth cells were suspended in a 1% agarose solution before implantation. Following injection of 10(6) cells into the frontal lobe of adult CBA or AKR mice, discrete tumor masses greater than 4 mm in diameter were obtained in 90% of animals at 14 days, and the largest tumors in adult mice occurred between 21 and 28 days after implantation. The tumor size following implantation of 10(6) cells was significantly greater than with 10(5) cells at 7 days (P less than 0.05) and at 14 and 21 days (P less than 0.01). Less than 60% of mice of BALB/c, RIII, or C57 black strains developed tumors greater than 4 mm diameter at 14 days after intracerebral injection of 10(6) C6 cells. Using neonatal mice it was found that when 10(5) cells were injected intracranially tumors greater than 4 mm in diameter developed in 14 of 15 animals within 2 weeks (CBA mice). Similar results were seen in the RIII, AKR, C57 black, and BALB/c strains. Longer growth periods resulted in larger tumors, up to 8 mm in diameter (6 of 10 animals at 20 days). The tumors in the neonatal animals were not as discrete as in the adult mice, and tumor often spread to the meninges and into the lateral ventricles. The tumor harvested from the brain had a cloning efficiency of 1.2 +/- 0.4% (SD). A panel of monoclonal antibodies was raised to the C6 glioma, and this was used to define clearly the margins of the tumor within the brain. The xenograft mouse models should prove useful for the study of the therapy of gliomas.

Nature of the tumor-localizing components of hematoporphyrin derivative.

Hematoporphyrin derivative linked by ether bonds (HE) was synthesized by unambiguous procedures in order to compare its properties to hematoporphyrin derivative (HpD). Reverse phase high performance liquid and gel filtration chromatography were used to compare the HE derivatives to HpD. The cellular uptake of HE derivatives was compared to HpD using the WEHI 3B (D+) cell line and was shown to be taken up to a degree and in a manner similar to HpD. The efficiency of HE porphyrins as photosensitizers was compared to HpD using the V79 cell line. HE porphyrins were more efficient in sensitizing the V79 cells than was HpD. The in vivo tumor localizing properties of HE porphyrins were compared to HpD in CBA mice bearing the C6 cerebral glioma, and BALB/c mice bearing the EMT6 mammary tumor. HE derivatives localized in both tumor models as effectively as HpD. We conclude that the properties of ether linked hematoporphyrin derivatives are very similar to properties of HpD.

Effects of bombesin antagonists on the growth of small cell lung cancer cells in vitro.

Small cell lung cancer (SCLC) produces several neuroendocrine peptides, including gastrin-releasing peptide (GRP), the mammalian equivalent of bombesin. There is some evidence to support the suggestion that GRP is an autocrine regulator of SCLC growth. Therefore, we have tested the effect of bombesin and two antagonists of bombesin on SCLC cell growth in a serum-free liquid tissue culture system. The antagonists used were analogues of substance P: spantide and (D-Arg1, D-Pro2, D-Trp7,9, Leu11) substance P. The cell lines used in this study all produced GRP-related peptides and one line had demonstrable GRP receptors. Exogenous bombesin did not cause any stimulation of growth in the liquid culture assay. The bombesin antagonists inhibited SCLC cell growth, but apparently not via the bombesin receptor. The bombesin used was biologically active because it stimulated the proliferation of Swiss 3T3 fibroblasts. The antagonists caused inhibition of this bombesin-induced proliferation, which was reversed by addition of excess bombesin. In addition, the antagonists and substance P alone stimulated proliferation of 3T3 cells, indicating that they may interact with another growth factor receptor on 3T3 cells. We conclude that growth of SCLC cells is not dependent on bombesin under all in vitro culture conditions because bombesin failed to stimulate growth in liquid cultures and the growth inhibition caused by bombesin antagonists was probably not mediated by the bombesin receptor.

Heterogeneous cytogenetic abnormalities in small cell lung cancer cell lines.

Ten cell lines were established from different biopsies from nine patients with small cell lung cancer (SCLC). These were established from metastases in the marrow (7), breast (1), pleural fluid (1), and spinal cord (1) and had been in culture for periods varying from 1 to 11 months. All cell lines exhibited typical cytological features of SCLC, and produced neuron specific enolase. All lines examined (5) contained dense core granules and were tumorigenic when injected intracranially into nude mice. None of the six cell lines tested had an amplified oncogene (c-myc or n-myc) previously reported to be amplified in SCLC. Detailed chromosome analyses were undertaken and showed that a structural abnormality of chromosome 3(p11p23) was a frequent (6 of 10) but not invariable finding. Our study and one other indicate that there is not a unique chromosome abnormality present in all cases of SCLC, although loss of chromosome 13 and structural abnormalities of chromosome 3 were frequently found. A feature of these SCLC cell lines established from metastatic deposits was the complexity of the karyotypes due to numerical and structural chromosomal abnormalities.

Specific killing of human melanoma cells by 125I-labeled 9.2.27 monoclonal antibody.

The anti-melanoma antibody 9.2.27 localizes to melanoma cells when administered i.v. to melanoma patients, but high doses of this antibody alone have no specific cytotoxic effect in vivo. To determine whether radiolabeled antibodies would exhibit specific antimelanoma cytotoxicity in vitro, cell survival curves were established for NCl-N892 human melanoma cells treated with 125I-labeled 9.2.27 monoclonal antibody. The binding capacity per cell was 5 X 10(5) molecules of 9.2.27 immunoglobulin G, and the association constant of binding was 10(10) M-1. Antibody preparations with specific radioactivities of 9-80 microCi/micrograms were used. Colony-forming ability after in vitro exposure to 125I-9.2.27 was determined by a 1-h antibody incubation at saturating concentrations, washing, and cell freezing for various exposure durations. Colony survival was dose dependent, varying with the radioactivity per cell and the exposure time. The survival curves demonstrated no shoulder effect and had a 37% incremental survival dose of 0.5-0.9 X 10(5) decays/cell. Selective killing of melanoma cells was demonstrated in experiments where NCl-N417 lung cancer cells were mixed with the melanoma cells prior to antibody treatment. The NCl-N417 cells did not express the melanoma-associated antigen, were more sensitive to conventional external irradiation than were the melanoma cells, and could easily be distinguished from them by different growth morphology. In spite of a growth advantage for the melanoma cells in the clonogenic assay, the antigen-negative lung cancer cells selectively survived the treatment and were the only surviving cells after 15 days of exposure.

Fluctuations in the expression of a glycolipid antigen associated with differentiation of melanoma cells monitored by a monoclonal antibody, Leo Mel 3.

A monoclonal antibody, Leo Mel 3, raised against a melanoma cell line (LiBr), binds to a carbohydrate determinant of cell surface gangliosides, the simplest of which is GD3. This monoclonal antibody was screened for by its capacity to block the recognition and lysis of the melanoma cells by cytotoxic T-lymphocytes with anomalous killer cell function, illustrating a novel approach for identifying monoclonal antibody to biologically relevant tumor-associated antigens. Leo Mel 3 reacted selectively with melanoma cells by indirect immunofluorescent and immunoperoxidase staining; it reacted with tissue from all primary and metastatic melanoma tested, and it bound to cells from all but one of six cultured melanoma cell lines. Leo Mel 3 did not react with a variety of carcinomas, lymphomas, leukemias, and other neuroectodermal tumors, nor with adult or fetal tissues, except fetal liver. Very weak staining of cutaneous basal melanocytes was noted in a minority of skin sections, and 50 to 80% of melanocytes in four of seven benign nevi showed weak to moderate reactivity. The antibody was relatively specific for human adherent melanoma cells, since it did not bind to the adherent murine B16 melanoma line nor to a nonadherent human melanoma cell line (PMC-22). Expression of the Leo Mel 3-defined antigen was unrelated to changes in cell cycle. When cells from an adherent melanoma cell line were detached and maintained briefly in suspension culture, the cells became markedly less reactive with Leo Mel 3 and, after readherence to plastic, they rapidly reexpressed higher levels of the ganglioside antigen; since Leo Mel 3 prevented attachment and growth of melanoma cells in vitro, a functional role for the ganglioside is suggested in cell adhesion and metastasis. Differentiation of melanoma cells with dimethyl sulfoxide, retinoic acid, and theophylline resulted in a marked and selective increase in the amount of Leo Mel 3-defined antigen, together with an increase in the target cell binding ability of these cells, assessed by cold target competition assays using anomalous killer cells.

Pharmacokinetics and toxicity of two schedules of high dose epirubicin.

Epirubicin, a stereoisomer of doxorubicin, is reported to have equal antitumor activity with lower cardiac and systemic toxicity. Recently the maximum tolerated dose of this drug has been revised upwards with reported increased response rates. However, the pharmacokinetics of epirubicin at high doses have never been reported. Accordingly, this study was designed to evaluate the pharmacokinetics of epirubicin when administered as either a 15-min i.v. bolus or a 6-h i.v. infusion in a phase I study at high doses. Nineteen patients with a variety of malignancies were given a total of 52 cycles of epirubicin at doses of 90 to 150 mg/m2 given once every 3 weeks. The maximum tolerated dose was 150 mg/m2 epirubicin given either as a bolus or as an infusion. The major dose-limiting toxicity was neutropenia. Interpatient variation occurred in the pharmacokinetics at each dose level but overall there were dose-dependent pharmacokinetics. This was manifested as a disproportionate increase in plasma levels and areas under the curve as the epirubicin dose was increased from 90 to 150 mg/m2. The pharmacokinetics of epirubicin could best be described by an open two-compartment model. Peak plasma concentrations were attained at a median of 12 min following the bolus injection and concentrations approached the steady state within a median of 55 min following the start of the 6-h infusion. Administration of the 150 mg/m2 dose over the 6 h compared to the bolus administration was associated with a 92% decrease in peak concentration from 3088 +/- 1503 to 234 +/- 126 ng/ml. This was not associated with an appreciable change in hematological or nonhematological toxicities. The median distribution half-life was 10 min and the median elimination half-life was 42.0 h. The cumulative renal excretion of the parent compound accounted for less than 2% of the administered dose. The major metabolites in both plasma and urine samples were 4'-O-beta-D-glucuronyl-4'-epidoxorubicin, 13-S-dihydro-4'-epidoxorubicin, and 4'-O-beta-D-glucuronyl-13-S-dihydro-4'-epidoxorubicin. This study demonstrates that a 135 mg/m2 bolus infusion given on a 3-weekly schedule is an appropriate initial dose for further clinical studies.

Carboplatin (CBDCA, JM-8) and VP-16-213 in previously untreated patients with small-cell lung cancer.

The efficacy and toxicity of carboplatin 100 mg/m2, administered intravenously (IV) daily X 3, and VP-16-213 120 mg/m2, IV daily X 3, administered every 28 days for six courses, was assessed in 94 (36 limited stage, 58 extensive stage) previously untreated patients with small-cell lung cancer. Mediastinal irradiation using 50 Gy in 25 fractions was given to all limited-stage patients with a complete (CR) or partial response (PR) after three chemotherapy courses. Cranial irradiation was administered to all patients with CR. Objective responses were seen in 77% (CR 40%, PR 37%) of patients with limited-stage and 58% (CR, 9%; PR, 49%) with extensive-stage disease. Median relapse-free survival for objective responders with limited stage was 14.6 months and 7.9 months for extensive-stage patients. Median relapse-free survival following CR was 15.4 months and 8.5 months for PR. Median survival was 15.3 months for limited-stage and 8.1 months for extensive-stage patients. The combination was well tolerated with mild nausea or less (World Health Organization [WHO] grade 0 or 1) in 62% of patients and minimal mucositis, renal, neurotoxicity, or ototoxicity. Neutropenia less than 1.0 X 10(9)/L (WHO grade 3 or 4) was seen in 63% of patients, with two deaths from infection while neutropenic. The combination of carboplatin and VP-16-213 is a new, active program with low toxicity when applied intensively in previously untreated patients with small-cell lung cancer.

Improved survival in esophageal cancer in the period 1978 to 1983.

The outcome of therapy of adenocarcinomas and squamous-cell carcinomas of the esophagus is so poor that the results of new approaches to therapy, such as the addition of radiotherapy or chemotherapy, are often compared with those achieved in historical controls. To determine the validity of this approach in cancers with a poor outcome, the results of therapy were analyzed at our institution from Jan 11, 1978 to Aug 9, 1981 (77 patients) and compared with the results achieved in the period from Aug 14, 1981 to Feb 19, 1984 (77 patients). The patients were evenly matched for prognostic factors. It was found that the median survival of the first group of patients (4 months) was significantly less (P less than .01) than that of the recently treated group (10 months). This was due to the better median survival of patients treated surgically from 4 months in the early group to 29 months at present (P less than .01). There was no change in the survival of the other patients. The major improvement in the outcome of surgery was due to the reduction of the perioperative mortality rate to less than 5%. There was no detectable change in patient selection for surgery. The results indicate that even in tumors with a very poor outcome, such as esophageal cancer, large changes in results can occur without a specific change in therapy, and that historical controls may be misleading.

Continuous intravenous infusions of bromodeoxyuridine as a clinical radiosensitizer.

Twelve patients were treated with continuous intravenous (24-hour) infusions of bromodeoxyuridine (BUdR) at 650 or 1,000 mg/m2/d for up to two weeks. Myelosuppression, especially thrombocytopenia, was the major systemic toxicity and limited the infusion period to nine to 14 days. However, bone marrow recovery occurred within seven to ten days, allowing for a second infusion in most patients. Local toxicity (within the radiation field) was minimal, with the exception of one of four patients, who underwent abdominal irradiation. Pharmacology studies revealed a steady-state arterial plasma level of 6 X 10(-7) mol/L and 1 X 10(-6) mol/L during infusion of 650 and 1,000 mg/m2/d, respectively. In vivo BUdR uptake into normal bone marrow was evaluated in two patients by comparison of preinfusion and postinfusion in vitro radiation survival curves of marrow CFUc with enhancement ratios (D0-pre/D0-post) of 1.8 (with 650 mg/m2/d) and 2.5 (with 1,000 mg/m2/d). In vivo BUdR incorporation into normal skin and tumor cells using an anti-BUdR monoclonal antibody and immunohistochemistry was demonstrated in biopsies from three patients revealing substantially less cellular incorporation into normal skin (less than 10%) compared with tumor (up to 50% to 70%). We conclude that local and systemic toxicity of continuous infusion of BUdR at 1,000 mg/m2/d for approximately two weeks is tolerable. The observed normal tissue toxicity is comparable with our previous clinical experience with intermittent (12 hours every day for two weeks) infusions of BUdR. Theoretically, a constant infusion should allow for greater incorporation of BUdR into cycling tumor cells and thus, for further enhancement of radiosensitization.

Treatment of chemotherapy-induced neutropenia by subcutaneously administered granulocyte colony-stimulating factor with optimization of dose and duration of therapy.

In patients who have not received extensive prior chemotherapy or radiotherapy, it has been previously demonstrated that granulocyte colony-stimulating factor (G-CSF) abrogated the leukopenia following administration of melphalan (25 mg/m2). This study examined the necessity of a prechemotherapy period of G-CSF administration and the effect of varying the timing and duration of postchemotherapy G-CSF. Initially, patients received 0.3, 1.0, 3.0, and 10 micrograms/kg/d subcutaneously on days 1 to 5 and days 10 to 18. Melphalan was given on day 9. In the next portion of the study melphalan was administered on day 1 and G-CSF, 10 micrograms/kg/d, was administered by subcutaneous infusion on five schedules: (1) days 2 to 13; (2) days 8 to 13; (3) days 2 to 18; (4) days 8 to 18; (5) days -9 to -2 and 2 to 13. G-CSF produced a rapid and sustained elevation in neutrophil levels within 24 hours even when started 8 days after melphalan. This treatment was sufficient to abrogate the neutropenia in patients who had received no prior chemotherapy. It was not necessary to continue G-CSF for more than 7 days. G-CSF did not consistently alter the course of the thrombocytopenia that followed this dose of melphalan. G-CSF was well tolerated, although mild bone pain occurred and was reduced with acetaminophen. One of 22 patients developed cellulitis at an infusion site. We conclude that after melphalan chemotherapy, G-CSF may need to be given for only a short period to prevent chemotherapy-induced neutropenia, and that G-CSF induces a rapid rise in neutrophil levels even when started 8 days after melphalan administration.

Pharmacology of the colony-stimulating factors.

Leukocyte production is influenced by a family of glycoproteins called colony-stimulating factors. Two of these have been purified, cloned and produced in quantities sufficient for clinical use. Granulocyte colony-stimulating factor (G-CSF) preferentially stimulates neutrophil production and has been shown to reduce the duration of neutropenia following chemotherapy. G-CSF therapy also has beneficial effects in a variety of other neutropenic states. Granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulates neutrophil, monocyte and eosinophil production and function. GM-CSF is associated with more diverse haematological and clinical effects. George Morstyn and colleagues summarize the promising results from the early clinical trials with these new therapeutic agents.

In vivo incorporation of bromodeoxyuridine into proliferating cells in the marrow and its effects on granulocyte-macrophage progenitor cells.

Bromodeoxyuridine (BUdR), a potential radiosensitizing drug, was given by intravenous infusion at 650-1000 mg/m2/day for up to 12 days. In vivo incorporation into human bone marrow was assayed by differential chromatid staining as well as by comparison of in vitro radiation survival curves of granulocyte-macrophage progenitor cells scored at both day 7 and day 14. Although a difference was found in the radiation survival of control (untreated) day-7 progenitor cells (Do = 1.39 Gy) and day-14 progenitor cells (Do = 0.89 Gy), a similar degree of in vitro radiosensitization was found for BUdR-treated bone marrow progenitor cells scored at day 7 and day 14. The culture technique provided a bioassay for the in vivo action of BUdR. BUdR treatment produced transient moderate myelosuppression that probably resulted from BUdR incorporation into normal marrow cells.

Clinical experience with recombinant human granulocyte colony-stimulating factor and granulocyte macrophage colony-stimulating factor.

Bacterially synthesized human granulocyte colony-stimulating factor (G-CSF) and granulocyte macrophage colony-stimulating factor (GM-CSF) have been studied to determine if they could prevent or reduce the neutropenia caused by chemotherapy. Our studies suggest that 10 micrograms/kg/day of G-CSF administered as a continuous subcutaneous infusion abrogates the neutropenia associated with a standard dose of melphalan. G-CSF produced a rapid increase of neutrophil levels, was well-tolerated, and was associated with only one frequent adverse effect: bone pain. GM-CSF, administered in doses ranging from 3 to 15 micrograms/kg/day subcutaneously, appeared to be useful in abrogating the neutropenia associated with chemotherapy, producing elevations in neutrophils, eosinophils, and monocytes. Although GM-CSF was relatively well-tolerated, several adverse effects, including bone pain, rashes, and fluid retention, were observed. The initial dose of GM-CSF in some patients produced a reaction that was characterized by hypoxia.

The effects of dose and route of administration on the pharmacokinetics of granulocyte-macrophage colony-stimulating factor.

The pharmacokinetics of granulocyte-macrophage colony stimulating factor (GM-CSF) (0.3-30 micrograms/kg) were studied after subcutaneous bolus (n = 16) or intravenous bolus (n = 5) injection or 2 h intravenous infusion (n = 12). Each method of administration gave a different GM-CSF concentration-time profile. Highest peak serum concentrations (Cmax) followed the intravenous bolus, and the time GM-CSF persisted at a concentration greater than 1 ng/ml (t greater than 1 ng/ml) was longer after a subcutaneous than after an intravenous injection. Area under the concentration-time curve (AUC), Cmax and t greater than 1 ng/ml all increased with dose for each method of administration. After intravenous administration, there was a two-phase decline in concentration. The half-life (t1/2) of the terminal phase following an intravenous bolus ranged from 0.24 to 1.18 h and, following intravenous infusion, from 0.62 to 9.07 h and appeared to increase with dose. The apparent clearance was greatest following subcutaneous injection at doses below 3 micrograms/kg, suggesting a saturable mechanism or different bioavailability. Only 0.001%-0.2% of the injected dose appeared in the urine as immunoreactive GM-CSF.

Characterisation of a cell line (LCC-18) from a cultured human neuroendocrine-differentiated colonic carcinoma.

A cell line (LCC-18) from a neuroendocrine colonic tumour was established. The tumour cells retained their endocrine characteristics through more than 100 passages and showed positive immunocytochemistry for synaptophysin, vasoactive intestinal polypeptide (VIP) and glucagon. The culture medium also contained VIP and glucagon, which indicates that mechanisms for release of some of the active peptides were preserved. Transplantation of LCC-18 tumour cells into nude rats resulted in tumour formation with similar endocrine characteristics. The c-myc gene was amplified which might have been a prerequisite for establishment of the cell line. The chromosomes in LCC-18 were studied by G-banding and C-banding. The cell line had a distinctive mode in the hypotriploid region, at S = 61. The double minute (Dms) positive stemline karyotype showed numerical and structural aberrations more similar to findings in ordinary colonic adenocarcinomas than to observations in previously studied, pure intestinal neuroendocrine tumours. The Dms may be correlated with amplification of c-myc. LCC-18 may become valuable for studies of neuroendocrine differentiation, regulation of growth and production and release of hormones and for studies of drug effect.