DNA-replication/DNA-damage-dependent centrosome inactivation in Drosophila embryos.

During early embryogenesis of Drosophila melanogaster, mutations in the DNA-replication checkpoint lead to chromosome-segregation failures. Here we show that these segregation failures are associated with the assembly of an anastral microtubule spindle, a mitosis-specific loss of centrosome function, and dissociation of several components of the gamma-tubulin ring complex from a core centrosomal structure. The DNA-replication inhibitor aphidicolin and DNA-damaging agents trigger identical mitotic defects in wild-type embryos, indicating that centrosome inactivation is a checkpoint-independent and mitosis-specific response to damaged or incompletely replicated DNA. We propose that centrosome inactivation is part of a damage-control system that blocks chromosome segregation when replication/damage checkpoint control fails.

Distinct enzymatic responses in mice exposed to a range of low doses of ozone.

Short-term exposure of mice to low O3 doses, as defined by the product of concentration and exposure time (ct), was observed to induce alterations in two enzyme systems: first, that leading to changes in hepatic reduced ascorbic acid (RAA) content, and second to changes in plasma creatine phosphokinase (CPK) activity. RAA alterations were noticed immediately, 30 min and 120 min after termination of the exposure period, whereas CPK showed alterations immediately and 15 min after termination of the exposure. Later determinations, i.e., 24 hr after O3 exposure for RAA and 30 min after 03 exposure for CPK, revealed no significant differences when compared to control animals. Although differences in sensitivity existed, the dose response curves for both systems were more or less similar, showing a short decrease for the initial very low O3 doses, followed by a profound rise and a gradual decrease to control levels for subsequent ct doses. Exceptions were the 30 min curve for RAA and the immediate curve for CPK in so far as that both showed an additional depression. Neither plasma histamine nor plasma lactic acid dehydrogenase (LDH3) were observed to be altered by the range of O3 doses employed. These findings were explained on the basis of adaptation of the organism to a potentially noxious O3 stimulus by enhanced metabolic processes: a weak stimulus leading to only a small adjustment, and stronger stimuli to elevated enzyme activity as well. With increasing doses of O3 this elevation in enzyme activity was found to be gradually diminished, possibly due to a steadily growing demand, leaving the overshoot becoming continually smaller until a balanced state is achieved.

Cytogenetic characterization of a high-grade murine B-cell lymphoma.

The chromosomal pattern of a high-grade malignant B-cell lymphoma that arose spontaneously in a C57BL mouse is described. A considerable amount of structural chromosomal abnormalities was found in the lymphoma cells. These abnormalities are discussed in view of their possible role for oncogenesis, infiltration, and tissue distribution. The chromosomal findings of this lymphoma are compared with the few that have been described previously.

Different homing pattern of isolated mouse lymphoma cells correlates with a different chromosomal pattern.

After 5-20 weeks of in vitro culture of mouse lymphoma cells, a characteristic and reproducible change in cell morphology, clonogenic ability, and homing pattern after intraperitoneal or intravenous injection was observed. Cytogenetic comparison of the two cell populations present before and after the "switch" revealed that the phenotypic changes cannot be due to in vitro karyotype evolution because their chromosomal pattern differed in such a way that it is impossible that they can evolve from each other. It was concluded that two different cell populations are present in the lymphoma and their growth and behavior are influenced by certain circumstances and/or interactions. Apparently one population predominates in the peripheral blood circulation, whereas the other will predominate after prolonged in vitro culturing.

Uptake of serotonin liberated by irradiation of rabbits and mice.

The radiation-induced release of serotonin (5-HT) in rabbits and mice has a linear dose-response relationship over a short range of approximately 0.75 Gy. In both animal species a threshold value is present, which is 1 Gy for rabbits and 5 Gy for mice. Doses higher than those falling in the linear range give rise to lower plasma 5-HT values. These lower values occur concurrent with increased splenic 5-HT levels. Stimulated 5-HT uptake of the spleen may be responsible for the diminished increase in plasma 5-HT.

Cisplatin sensitivity and thermochemosensitisation in thermotolerant cDDP-sensitive and -resistant cell lines.

Development of thermotolerance is an important phenomenon that must be considered when thermochemotherapy with multiple heat treatments is used clinically. To study the effect of thermotolerance on cellular cisplatin (cDDP) sensitivity at 37 degrees C and 43 degrees C in cell lines with different cDDP sensitivities, two Ehrlich ascites tumour cell lines (one with high cDDP sensitivity and one with in vitro acquired cDDP resistance) were used. The results indicate that in both cell lines the state of thermotolerance per se did not affect the cDDP sensitivity at 37 degrees C. Thus, general elevations in 'all' heat shock protein levels as found in thermotolerant cells apparently do not influence cDDP sensitivity to a considerable extent. The sensitising effect of a (second) heat treatment given simultaneously with a cDDP treatment was less in thermotolerant cells. Thermal enhancement ratios (TERs) at the 10% survival level for heat doses of 43 degrees C for 30 min or 43 degrees C for 60 min were reduced by a factor of 1.6 and 2.1 in cDDP-resistant and -sensitive thermotolerant cells respectively, as compared with control cells. Thus, protection against heat damage in thermotolerant cells seems to be paralleled by diminished thermal chemosensitisation. Although the effect of thermotolerance on the cDDP-sensitising effect was less pronounced in the resistant cells, a modifying effect on the resistance factor was not achieved.

Enhanced cytostatic activity of the sesquiterpene lactone eupatoriopicrin by glutathione depletion.

Eupatoriopicrin (EUP), a sesquiterpene lactone from Eupatorium cannabinum L., possesses cytostatic activity. This was demonstrated for FIO 26 cells in vitro with the aid of a clonogenic assay and in vivo by tumour growth delay in FIO 26 and Lewis lung tumour-bearing mice. In vitro the IC50 for 1 h exposure to EUP was 1.5 microgram ml-1 (4.1 nmol ml-1). This concentration depleted about 25% of its cellular GSH concentration. Pretreatment of FIO 26 cells with BSO, resulting in greater than 99%. GSH depletion, enhanced the cytotoxic effect of EUP. The dose-enhancement factor at the level of 10% cell survival was 2.3. Growth inhibition of the Lewis lung carcinoma and the FIO 26 fibrosarcoma, solidly growing in C57Bl mice, was found after i.v. injection of 20 or 40 mg kg-1 EUP, at a tumour volume of about 500 microliters. Pretreatment with BSO at a dose of 4 mmol kg-1 i.p., 6 h before EUP administration, resulted in a significantly stronger growth delay of both tumours compared with EUP only. At the time of EUP treatment, cellular GSH in the tumours was reduced by BSO treatment to about 60%. It is concluded that EUP possesses antitumour activity in vivo and that chemosensitisation of EUP may be accomplished by pretreatment with BSO, indicating that endogenous GSH protects against the cytostatic action of EUP.

Sensitizing for cis-diamminedichloroplatinum(II) action by hyperthermia in resistant cells.

cDDP-resistant Ehrlich ascites tumour (EAT) cells (ER cells) were tested for cellular content of total glutathione, heat sensitivity, cDDP sensitivity and synergistic effects of a combined treatment of heat and chemotherapy. In comparison with the non-resistant EAT cells (EN) the ER cells had an elevated level of glutathione. Treatment with D,L-buthionine-(S,R)-sulphoximine (BSO), resulting in almost complete depletion of cellular glutathione, did not cause drug sensitization. The ER cells were somewhat less heat sensitive compared with the EN cells. Heat chemosensitization was observed for the EN cells as well as for the ER cells. At 43 degrees C (but not at 42 degrees C) the thermal enhancement ratio (TER) for cDDP toxicity was significantly higher in the ER cells. The total number of cells killed by the combined treatment was less in the ER cells than in the EN cells. After analysing existing literature, combined with the current results, it is concluded that although cDDP-resistant cells can often considerably be chemosensitized by hyperthermia, in most cases the difference in cDDP sensitivity cannot be overcome totally. In those situations where cDDP-resistant cells are more sensitive to heat and also show a high TER, especially at clinically relevant temperatures, hyperthermia as added modality is indicated for clinical treatment.

Non-effectivity of roentgen irradiation in an in vivo rapidly growing murine lymphosarcoma.

The survival time of mice bearing a rapidly growing murine lymphosarcoma (cell doubling time 9.6 h) is hardly influenced by roentgen irradiation with fractions of 2 Gy, not even if delivered according to superfractionated schedules. Growth kinetic measurements reveal an almost complete mitotic block 6 h after exposure, followed by a large overshoot in cell proliferative activity. The dose-response curve for in vivo irradiated cells has a D0 of 1.9 and lacks a shoulder. Hence, repair of sublethal damage must be of restricted significance. The extremely strong proliferative capability appears to lead to rapid repopulation of the tumour. Typically, surviving cells show a high content of RNA and protein.

Suitability of rodent tumor models for experimental PET with L-[1-11C]tyrosine and 2-[18F]fluoro-2-deoxy-D-glucose.

The applicability of five different rodent tumors for experimental PET has been investigated. L-[1-11C]Tyrosine was a better indicator for the growth activity of the tumors than [18F]FDG. For experimental PET, the three mice models studied appeared inappropriate; the Lewis lung tumor and the fibrosarcomateous FIO 26 had too low a tyrosine utilization, while the lymphosarcomateous LY showed insufficient tumor-to-background ratios. Of the two rat models, the necrotic Walker 256 carcinosarcoma was less suitable. By using L-[1-11C]tyrosine, the solid, rhabdomyosarcoma tumor offers good possibilities of monitoring therapeutic interventions with PET.

Sensitization to cisplatin action by step-down heating in cDDP-sensitive and -resistant cells.

Hyperthermia treatment (> or = 43 degrees C) has been shown to be able to (partially) reverse acquired cDDP resistance. However, such heat treatment is difficult to achieve in the clinic. Short pre-treatment at a high temperature (> 42 degrees C), immediately before a treatment at a lower temperature (< 42 degrees C) can enhance the heat toxicity of the lower temperatures. This "step-down heating schedule" was explored for its possible drug-sensitizing potential in in vitro-cultured cDDP-sensitive and -resistant murine and human tumour cells. A 10-min pre-treatment at 44 degrees C enhanced the cytotoxicity of 41 degrees C hyperthermia alone. It also enhanced sensitivity to cDDP when given at 37 degrees C. However, it did not increase the 41 degrees C-induced cDDP sensitization. Thus, no correlation was found between heat kill and cDDP sensitization for step-down heating schedules. The observed effects of step-down heating were comparable in sensitive and in resistant cells, so the step-down heating schedule, unlike the 43 degrees C treatment, did not lead to a decrease of the cDDP-resistance factor. Yet the total cytotoxicity caused by this treatment protocol was 10-fold more than for cDDP with 41 degrees C alone, due to the extra hyperthermic cell killing and the cDDP-sensitizing effect of the pre-treatment. This treatment could have a substantial impact on cDDP efficacy in the clinic even when cDDP resistance has developed.

Heat-shock protein expression in cisplatin-sensitive and -resistant human tumor cells.

It has been suggested that the expression of certain heat-shock proteins (HSPs) may be prognostic markers in several tumor types. Since HSPs may be involved in determining cellular sensitivity to chemotherapeutic drugs, the possible relation between HSP expression and cisplatin (cDDP) sensitivity was studied. Three human germ-cell tumor cell lines, 1 human small-cell lung carcinoma (SCLC) cell line and 3 human colon carcinoma cell lines were used as a model for differences in intrinsic cDDP sensitivity. The constitutive expression of a panel of HSPs was studied by immunoblotting. No correlation was found between expression of HSP90, HSP73, HSP72, HSP60 and HSP27 and the extent of intrinsic cDDP sensitivity when all cell lines studied were considered. However, for the 3 cell lines derived from germ-cell carcinomas, HSP27 expression was inversely related to cDDP sensitivity; ie. decreased HSP27 levels were associated with decreased sensitivity. Constitutive HSP expression was also studied in 2 sets of human cell lines with in vitro acquired cDDP resistance. In both resistant cell lines, decreased expression of HSP27 (as determined by Western blotting) was found as compared to the sensitive parent cell lines. Thus, acquired resistance to cDDP was also accompanied by decreased HSP27 expression. Interestingly, when basal HSP27 mRNA levels were measured in the SCLC cell line (GLC4) and its subline with acquired resistance (GLC4-cDDP), no significant differences were detected. Continuous cDDP incubation increased HSP27 levels and induced HSP27 phosphorylation in GLC4 cells, but not in the resistant subline. Thus, although no general relationships between HSP expression and cDDP sensitivity are apparent, high HSP27 expression in vitro relates to high sensitivity to cDDP treatment in some tumor types. This is in accordance with reported clinical data on high HSP27 levels in tumors correlating with good prognosis.

Mechanism of hyperthermic potentiation of cisplatin action in cisplatin-sensitive and -resistant tumour cells.

In this study, the mechanism(s) by which heat increases cis-diamminedichloroplatinum (cisplatin, cDDP) sensitivity in cDDP-sensitive and -resistant cell lines of murine as well as human origin were investigated. Heating cells at 43 degrees C during cDDP exposure was found to increase drug accumulation significantly in the cDDP-resistant cell lines but had little effect on drug accumulation in the cDDP-sensitive cell lines. DNA adduct formation, however, was significantly increased in all cell lines studied. Furthermore, ongoing formation of platinum (Pt)-DNA adducts after the end of cDDP treatment was enhanced and/or adduct removal was decreased in heated cells, resulting in relatively more DNA damage remaining at 24 h after the end of cDDP exposure. Correlation plots with survival revealed weak correlations with cellular Pt accumulation (r2 = 0.59) and initial Pt-DNA adduct formation (r2 = 0.64). Strong correlations, however, were found with Pt-DNA adducts at 6 h (r2 = 0.97) and 24 h (r2 = 0.89) after the incubation with the drug. In conclusion, the mechanism by which heat sensitizes cells for cDDP action seems to be the sum of multiple factors, which comprise heat effects on accumulation, adduct formation and adduct processing. This mechanism did not seem to differ between cDDP-sensitive and -resistant cells, emphasizing the potential of hyperthermia to reduce cDDP resistance.

Hyperthermic potentiation of cisplatin toxicity in a human small cell lung carcinoma cell line and a cisplatin resistant subline.

A human small cell lung carcinoma cell line (GLC4) and its subline with in vitro acquired cisplatin (cDDP) resistance (GLC4-cDDP) were used to study the applicability of hyperthermia to interfere with acquired cDDP resistance. GLC4 and GLC4-cDDP did not differ in heat sensitivity (clonogenic ability). Both cell lines could be sensitized to cisplatin to a considerable extent, both at 42 and 43 degrees C. For 42 degrees C hyperthermia treatments up to 90 min no differences in TER between the cell lines were observed. Only prolonged (> or = 45 min) exposures to 43 degrees C hyperthermia sensitized the resistant cell line to a greater extent than the parent cell line, resulting in a reduction of the resistance factor from 3.6 (at 37 degrees C) to 1.7 (60 min 43 degrees C). The finding in this human system that for treatments up to 90 min, 43 degrees C heat is more suitable than 42 degrees C heat to reduce cDDP resistance, is in accordance with earlier findings with murine cells (Konings et al. 1993). Effects of heat, cisplatin and combined treatments on cell killing were not only measured with the clonogenic assay, but also with the microculture tetrazolium method (MTT assay), an assay of potential use in the clinic for rapid screening of cells obtained from patients. The data with the latter assay were comparable to those obtained with the clonogenic assay. However, its applicability to measure thermo-chemosensitization is limited due to its inability to measure more than one log of cell killing.

Heat-shock-protein-27 (hsp27) expression in ovarian carcinoma: relation in response to chemotherapy and prognosis.

Heat-shock protein 27 (hsp27) is one of the small heat-shock proteins. Its expression in ovarian- and breast-cancer cell lines has been associated with resistance to cisplatin and doxorubicin. In addition, hsp27 expression appears to facilitate cellular growth, differentiation and motility. In several human carcinomas, hsp27 expression might also be related to worse prognosis. The aim of this study was to evaluate the prognostic value of hsp27 expression in patients with ovarian carcinoma in relation to their response to chemotherapy and overall survival. Hsp27 expression was assessed by immunohistochemistry in 77 patients with ovarian carcinoma stage IC-IV. All patients received cisplatin- and doxorubicin-based chemotherapy and had long-term follow-up. In 30 patients, paired tumour samples were available, obtained before and after chemotherapy. Hsp27 immunostaining was positive in 86% of patients before and in 72% of patients after chemotherapy. Hsp27 expression was not related to any clinicopathologic factor, including previously determined p53 expression. Univariate analysis showed that, in stage-III and -IV patients, younger age, no residual tumour after first laparotomy, < or = 1 litre ascites, response to first-line chemotherapy and absence of hsp27 expression were associated with longer median progression-free survival. However, in multivariate analysis, only age, ascites and response to chemotherapy retained independent prognostic value.