Age-dependent cell inactivation by vincristine alone or in combination with 1-propargyl-5-chloropyrimidin-2-one.

Inactivating effects caused by vincristine alone or in combination with another mitotic inhibitor, 1-propargyl-5-chloropyrimidin-2-one, were studied as loss of colony-forming ability in exponentially growing or synchronized populations of the human cell line NHIK 3025. Treatment with 4 ng vincristine per ml(4.3 nM) in G2 led to irreversible mitotic arrest. Both mitotic arrest and lethal damage due to vincristine were primarily induced when cells were exposed in late S and G2, suggesting a correlation between the cell cycle-inhibitory and inactivating effect of this drug at clinically relevant concentrations. No repair of sublethal damage after vincristine treatment could be detected within 5 hr. A common feature in the age response of NHIK 3025 cells to the two mitotic inhibitors is drug resistance in G1. However, while mitosis is the most sensitive stage in the cycle with respect to inactivation by 1-propargyl-5-chloropyrimidin-2-one, mitotic cells are relatively resistant to treatment with vincristine. The combined inactivating effect of vincristine and 1-propargyl-5-chloropyrimidin-2-one was purely additive during interphase. In mitosis, the two drugs demonstrated a striking synergistic effect.

Neuron-specific enolase as a prognostic factor in metastatic malignant melanoma.

Serum neuron-specific enolase (NSE) was measured in 63 patients with metastatic malignant melanoma. 20 patients (32%) had elevated serum NSE (> 10 micrograms/l) before the start of treatment. Another 13 patients (21%) developed pathological NSE values during the course of the disease. In many patients, elevated NSE was related to a large tumour burden, and a gradual rise in serum NSE indicated disease progression. Patients with elevated pretreatment NSE had a median survival time of 3 months compared with 12 months for those with normal pretreatment NSE values. NSE thus proved to be a useful prognostic factor in metastatic malignant melanoma.

Neuron specific enolase (NSE) in serum of patients with malignant melanoma.

Serial measurements of serum NSE were performed in 63 patients with metastatic melanoma. NSE was measured by a sensitive immunoradiometric assay based upon monoclonal anti-bodies with monodisperse magnetizable particles as the solid phase. Increased NSE values were found in 30 patients (48%) during the course of the disease. In 10 patients serum NSE normalized during systemic therapy. In 11 patients with initial normal serum NSE, the marker values became elevated as the tumor progressed.

Steroids and growth of a human cell line stemming from a carcinoma of the uterine cervix.

The effect of different steroids on the growth of a human cell line, was examined in NHIK 3025 cells, derived from a carcinoma of the uterine cervix. When this cell line was grown in Eagle's minimal essential medium (MEM) for 4 days, addition to the medium of estradiol-17 beta in concentrations ranging from 10(-9) to 10(-7) M did not promote significant cell growth. Stimulated growth was observed with testosterone in these concentrations, with maximal effect (29% above control) at 10(-7) M. 5alpha-Dihydrotestosterone over the range 10(-9)--10(-6) M gave no such effect. 4-Androstene-3,17-dione, 5alpha-androstan-3alpha,17beta-diol and 5alpha-androstan-3beta,17beta-diol had no significant effect on cell proliferation at a concentration of 10(-7) M, while 4-androstene-3beta,17beta-diol augmented cell number to values 50-60% higher than those of control cultures. The observed increase in cell number was due to a shortening of the cell cycle and to a higher plating efficiency. Thus, cell line NHIK 3025 has not the responsiveness to estradiol-17beta of normal uterine cells. The data suggest that testosterone and/or 4-androstene-3beta,17beta-diol may be growth factors for this cell line.

Recovery from x-ray induced damage in human cells grown in culture.

Cells from an established human cell-line, NHIK 3025, originally derived from an early stage of cancer of the cervix, were tested for recovery capacity when irradiated with X-rays under aerobic (equilibrated with air) and extremely hypoxic conditions (O2 content less than 4 ppm). The dose-response curve obtained under aerobic conditions had a D0-value of 130 rads and an extrapolation number of 3.8. The corresponding curve obtained with a first dose exceeding that of the shoulder region of the survival curve followed 3 hours later by graded second doses, had a D0-value of 130 rads and extrapolation number 2.2. The curve obtained by measuring the survival after two equal doses of 330 rads, each with variable time intervals between the doses, showed a time dependent radiosensitivity. The dose-response curve obtained by irradiating the cells under extremely hypoxic conditions was well fitted by an exponential line up to about 2500 rads followed by a downward bend. Recovery from sublethal damage was not observed in split-dose experiments where the total doses were less than 2500 rads. For a total dose of 4200 rads, however, a split-dose effect was observed (SDR = 1.6). This split-dose effect is probably not due to Elkind repair, but is rather a consequence of the technique used.

Inactivation by the mitotic inhibitor NY 3170 of human cells in vitro.

Inactivation of NHIK 3025 cells ny the mitotic inhibitor NY 3170 (1-propargyl-5-chloropyrimidin-2-one) was measured as loss of colony-forming ability. NY 3170 at a concentration of 0.15 nM allowed no formation of colonies after 12 days of continuous exposure to the drug. Metaphase arrest after treatment with NY 3170 was reversible if the drug was removed immediately after the onset of the arrest. When the cells were kept in mitosis by the presence of NY 3170, inactivation was complete after 8h incubation of mitotic cells with 0.4 nM NY 3170. Using synchronized cell populations, it was shown that mitosis is by far the most sensitive stage of the cell cycle to inactivation by NY 3170. This leads to the suggestion that there is a connection between the inactivating and the metaphase-arresting effect of this drug. The age response curves show that after mitosis the stages in order of decreasing sensitivity to NY3170 are: G2, late S, early S and G1. This is a similar age response to that reported for proliferating cells treated with bleomycin, whereas the mitotic inhibitors vincristine and vinblastine have shown qhite different age response curves.

Cell kinetic characteristics in different parts of multicellular spheroids of human origin.

The growth fraction, the cell cycle time, and the duration of the individual cell cycle phases were determined as a function of distance from the surface of multicellular spheroids of the human cell line NHIK 3025. The techniques employed were percentage of labelled mitoses and labelling index measurements after autoradiography and flow cytometric measurements of DNA histograms. To separate cell populations from the different parts of the spheroid, fractionated trypsinization was employed. The results were compared with corresponding values in NHIK 3025 cell populations grown as monolayer cultures. While practically all cells in exponentially growing monolayer populations are proliferating, the growth fraction was between 0.6 and 0.7 in the outer parts of the spheroid. The inner region was mainly occupied by a necrotic mass. The proliferating fraction of the recognizable cells in the inner region was slightly below 0.5. The mean cell cycle time of NHIK 3025 cells in monolayer culture is 18 hr. The mean cell cycle time of proliferating cells in the periphery of the spheroid was 30 hr, compared to 41 hr in the inner region (150 micrometer from the spheroid surface). All phases of the cell cycle were prolonged compared to populations of exponentially growing monolayer cells. Within each part of the spheroid the distribution of cell cycle times was considerably broadened compared with monolayer populations.

Cell-cycle inhibition by misonidazole of human cells cultivated in vitro under aerobic conditions.

By means of flow cytometric recording of DNA histograms and counting of cells in synchronized populations, we have found that misonidazole (MIS) in clinically relevant concentrations induces cell-kinetic changes in human cells (NHIK 3025) cultivated in vitro under aerobic conditions. The effect seems to be a general lengthening of the cell cycle, affecting all phases. However, induction of this effect is phase-dependent, since only cells exposed to MIS during mitosis and/or early G1 will suffer significant cell-cycle prolongation. In exponentially growing populations this effect of MIS leads to a transient increase in the fraction of G1 cells and a corresponding decrease in the fraction of S cells. The possible significance of this effect for the clinical use of MIS is discussed.

Photodynamic effect of haematoporphyrin throughout the cell cycle of the human cell line NHIK 3025 cultivated in vitro.

Cells from the established cell line NHIK 3025 were synchronized by repeated mitotic selections. Survival of the synchronized cells after treatment with haematoporphyrin and near-UV light was measured by testing the capacity of the cells to form macroscopic colonies. The sensitivity to photodynamic inactivation was small in early G1, late S and G2. The sensitivity increased throughout late G1 and early S to a maximum in mid S. More than a 100-fold variation is found in the survival after 20 min irradiation in the presence of 4 X 10(-4)M haematoporphyrin.

Cellular and humoral factors in host susceptibility to Lewis lung carcinoma.

Cellular and humoral anti-tumour reactivity in strains of mice highly susceptible (C57Bl/6) or less susceptible (C57Cl/6 x DBA/2 = B6D2F1) to Lewis lung carcinoma (LLC) was investigated. Natural killer cell activity in a 51Cr release assay against this tumour could be demonstrated with a good correlation to in vivo susceptibility. This has not been demonstrated earlier for solid, spontaneous tumours. T-cell deficiency (congenital athymic (nude mice)) did not affect the cumulative incidence of tumour take. However, the number of lung metastases was significantly reduced in nude mice. Treatment with antilymphocyte serum (ALS) increased the susceptibility to LLC in both strains. In a soft agar colony assay a marked reduction in the number of colonies was observed when tumour cells were incubated with serum from B6D2F1 mice as compared to serum from C57Bl/6 mice, prior to seeding. Apparently naturally occurring cellular, as well as humoral effector mechanisms are involved in host resistance to Lewis lung carcinoma in the mouse.

Steroid responsiveness of the human cell line NHIK 3025.

The human cell line NHIK 3025, derived from a carcinoma of the uterine cervix, contains a glucocorticoid and an androgen receptor. The effect of various natural and synthetic steroid hormones and antihormones on growth rate of these cells was therefore investigated. Cells grown in Eagle's MEM with 10% foetal calf serum exhibited reduced growth when cultured with dexamethasone due to prolongation of the cell cycle. Glucocorticoid anti-inducers like progesterone had no significant effect on cell growth. Methyltrienolone (R 1881) or 5 alpha-dihydrotestosterone did not affect cell proliferation. The reported shortening of the cell cycle by testosterone is probably not directly connected with activation of the androgen receptor present, but possibly dependent on metabolic conversion of testosterone to the more potent growth stimulator 4-androstene-3 beta, 17 beta-diol. The effect of several anti-androgens was also studied. The non-steroidal anti-androgens flutamide and SCH 16483 had no significant effect on cell proliferation. It was, however, found that a number of steroid anti-androgens, including R 2956, stimulated cell growth. A significant stimulatory effect by R 2956 was seen within the first cell generation, 4-androstene-3 beta, 17 beta-diol had to be present during 2 days, and testosterone for even longer times before a similar effect on cell growth could be obtained.

Multicellular spheroids grown directly from human tumour material.

Human tumour cells from surgical material were grown as multicellular spheroids. In 16 out of 20 cultures spheroids with a diameter of more than 250 microns could be observed. In 6 out of 20 cultures more than 30 spheroids with a diameter of at least 300 microns were obtained, i.e. 30% of the cultures fulfilled the criterion for a possible chemosensitivity test on primary cell spheroids. A study of stained sections from the spheroids and the respective tumours, showed that the morphology of the spheroid was very similar that of the tumour from which the cells were derived. Samples from 9 malignant melanomas, 4 bladder carcinomas, 2 renal-cell carcinomas, 2 ovarian carcinomas, 1 lymphoma, 1 colon carcinoma, and 1 schwannoma were tested for spheroid growth. Spheroids were obtained from at least one representative of all these tumour types. However, investigations involving larger numbers of tumours are needed to find out which tumour types are most suitable for further biological characterization of primary cell spheroids and tests for therapy response.

Cell-cycle inhibitory effects of the mitotic inhibitor NY 3170 on human cells in vitro.

Effects of the mitotic inhibitor NY 3170 (1-propargyl-5-chloropyrimidin-2-one) on cell-cycle kinetics of NHIK 3025 cells were studied by means of time-lapse microcinematography, pulsed incorporation of [3H] thymidine, flow cytometry, and mitotic index. All the experiments were performed with cells synchronized by mitotic selection. Mitotic inhibition as well as inhibition in interphase was examined. The small fraction of cells able to escape mitotic arrest at 0.2mM NY 3170 had spent about 12 h in metaphase. The metaphase block was complete at 0.3 mM. For comparison, complete metaphase arrest of NHIK 3025 cells was reached at 8 mM after treatment with the parent substance NY 3000 (5-chloropyrimidin-2-one, previously reported). At 0.3mM NY 3170 interphase was also considerably prolonged. All stages of interphase were prolonged, in contrast to the interphase prolongation after treatment with high concentrations of the mitotic inhibitors vincristine and vinblastine, which occurs in G2. It was shown that the presence of NY 3170 during mitosis is a necessary and sufficient condition for metaphase arrest, thus demonstrating that metaphase arrest is not dependent on some preceding event in interphase.

Effects of steroids and different culture media on cell cycle of the androgen-sensitive human cell line NHIK3025.

The median cell cycle of synchronized NHIK3025 cells grown in Eagle's MEM with 10% foetal calf serum was 23.6 h, compared to 18 h observed earlier in Puck's E2a medium with 30% serum (20% human and 10% horse). This difference is due to prolongation of both G1 and G2 in the MEM type medium. Testosterone and 4-androstene-3 beta, 17 beta-diol reduced the cell cycle of synchronized populations of NHIK3025 cultured in MEM type medium. Dexamethasone gave cell cycle prolongation while estradiol had no effect. These results are in accordance with steroid-induced changes in growth of asynchronous cell populations observed earlier. The androgen growth stimulation was partly due to a shortening of G2 but was not exclusively located in one particular phase of the cell cycle.

Inhibitory effect on tumour colony formation of mouse serum associated with tumour resistance in vivo in semi syngeneic mice.

Differences in tumour susceptibility between strains of mice (C57Bl/6 and C57Bl/6 X DBA/2 = B6D2F1) could be demonstrated for several tumours of C57Bl origin, both solid tumours (B16 melanoma and Lewis lung carcinoma) and lymphomas (RBL-5, 136-3 and ALC). Serum from mice with high tumour resistance in vivo (B6D2F1) showed an inhibitory effect on tumour colony formation in a soft agar colony assay. Serum from mice with lower tumour resistance (C57Bl/6) had no effect. When other F1 hybrids of C57Bl/6 parental origin were tested, the same correlation between in vitro inhibition of tumour colony formation and in vivo susceptibility was found. The serum factor was species non-specific, since the activity was expressed against in vitro grown cell lines of human origin. The tumour colony inhibitory activity was heat sensitive (56 degrees C for 30 minutes), precipitable by (NH4)2SO4, and not removed by adsorption on tumour cells. These results demonstrate the existance of a naturally-occurring humoral tumerostatic factor(s) which correlates to in vivo susceptibility to tumour cells. Its relationship to NK cell activity is discussed.

[Tumor lysis syndrome. A life-threatening complication during cytostatic treatment of chemosensitive types of cancer]. / Tumorlysesyndrom. En livstruende komplikasjon ved cellegiftbehandling av kjemosensitive kreftformer.

Tumour lysis syndrome is characterized by hyperkalemia, hypermagnesemia, hyperphosphatemia, hyperuricemia and elevated levels of serum creatinine and urea, indicating renal dysfunction due to rapid lysis of a large number of tumour cells. Hypocalcemia is often found as well. Tumour lysis syndrome is most likely to occur after chemotherapy of cancers that are highly responsive to chemotherapeutic drugs, such as malignant lymphomas and acute leukemias, when there is a large burden of tumours. Another risk factor is impaired renal function before treatment. Tumour lysis syndrome is potentially life-threatening. During the first course of chemotherapy in particular, the patient should receive prophylactic treatment, and laboratory tests should be performed daily in order to avoid this serious complication.