Willy Lemstra

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 γ-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.

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, ≤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. Int. J. Cancer (Pred. Oncol.) 84:234–238, 1999.

De novo CoA biosynthesis is required to maintain DNA integrity during development of the Drosophila nervous system

In a forward genetic screen in Drosophila melanogaster, aimed to identify genes required for normal locomotor function, we isolated dPPCS (the second enzyme of the Coenzyme A biosynthesis pathway). The entire Drosophila CoA synthesis route was dissected, annotated and additional CoA mutants were obtained (dPANK/fumble) or generated (dPPAT-DPCK). Drosophila CoA mutants suffer from neurodegeneration, altered lipid homeostasis and the larval brains display increased apoptosis. Also, de novo CoA biosynthesis is required to maintain DNA integrity during the development of the central nervous system. In humans, mutations in the PANK2 gene, the first enzyme in the CoA synthesis route, are associated with pantothenate kinase-associated neurodegeneration. Currently, the pathogenesis of this neurodegenerative disease is poorly understood. We provide the first comprehensive analysis of the physiological implications of mutations in the entire CoA biosynthesis route in an animal model system. Surprisingly, our findings reveal a major role of this conserved pathway in maintaining DNA and cellular integrity, explaining how impaired CoA synthesis during CNS development can elicit a neurodegenerative phenotype.

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.

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, i.e. 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.

Grp/DChk1 is required for G2-M checkpoint activation in Drosophila S2 cells, whereas Dmnk/DChk2 is dispensable.

Cell-cycle checkpoints are signal-transduction pathways required to maintain genomic stability in dividing cells. Previously, it was reported that two kinases essential for checkpoint signalling, Chk1 and Chk2 are structurally conserved. In contrast to yeast, Xenopus and mammals, the Chk1- and Chk2-dependent pathways in Drosophila are not understood in detail. Here, we report the function of these checkpoint kinases, referred to as Grp/DChk1 and Dmnk/DChk2 in Drosophila Schneiders cells, and identify an upstream regulator as well as downstream targets of Grp/DChk1. First, we demonstrate that S2 cells are a suitable model for G2/M checkpoint studies. S2 cells display Grp/DChk1-dependent and Dmnk/DChk2-independent cell-cycle-checkpoint activation in response to hydroxyurea and ionizing radiation. S2 cells depleted for Grp/DChk1 using RNA interference enter mitosis in the presence of impaired DNA integrity, resulting in prolonged mitosis and mitotic catastrophe. Grp/DChk1 is phosphorylated in a Mei-41/DATR-dependent manner in response to hydroxyurea and ionizing radiation, indicating that Mei-41/ATR is an upstream component in the Grp/DChk1 DNA replication and DNA-damage-response pathways. The level of Cdc25Stg and phosphorylation status of Cdc2 are modulated in a Grp/DChk1-dependent manner in response to hydroxyurea and irradiation, indicating that these cell-cycle regulators are downstream targets of the Grp/DChk1-dependent DNA replication and DNA-damage responses. By contrast, depletion of Dmnk/DChk2 by RNA interference had little effect on checkpoint responses to hydroxyurea and irradiation. We conclude that Grp/DChk1, and not Dmnk/DChk2, is the main effector kinase involved in G2/M checkpoint control in Drosophila cells.

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.

Contribution of growth and cell cycle checkpoints to radiation survival in Drosophila

Cell cycle checkpoints contribute to survival after exposure to ionizing radiation (IR) by arresting the cell cycle and permitting repair. As such, yeast and mammalian cells lacking checkpoints are more sensitive to killing by IR. We reported previously that Drosophila larvae mutant for grp (encoding a homolog of Chk1) survive IR as well as wild type despite being deficient in cell cycle checkpoints. This discrepancy could be due to differences either among species or between unicellular and multicellular systems. Here, we provide evidence that Grapes is needed for survival of Drosophila S2 cells after exposure to similar doses of IR, suggesting that multicellular organisms may utilize checkpoint-independent mechanisms to survive irradiation. The dispensability of checkpoints in multicellular organisms could be due to replacement of damaged cells by regeneration through increased nutritional uptake and compensatory proliferation. In support of this idea, we find that inhibition of nutritional uptake (by starvation or onset of pupariation) or inhibition of growth factor signaling and downstream targets (by mutations in cdk4, chico, or dmyc) reduced the radiation survival of larvae. Further, some of these treatments are more detrimental for grp mutants, suggesting that the need for compensatory proliferation is greater for checkpoint mutants. The difference in survival of grp and wild-type larvae allowed us to screen for small molecules that act as genotype-specific radiation sensitizers in a multicellular context. A pilot screen of a small molecule library from the National Cancer Institute yielded known and approved radio-sensitizing anticancer drugs. Since radiation is a common treatment option for human cancers, we propose that Drosophila may be used as an in vivo screening tool for genotype-specific drugs that enhance the effect of radiation therapy.

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.

Dysfunctional BRCA1 is only indirectly linked to multiple centrosomes

A remarkable and yet unexplained phenomenon in cancer cells is the presence of multiple centrosomes, organelles required for normal cell division. Previously, it was demonstrated that the tumor suppressor BRCA1 is a component of centrosomes. This observation led to the hypothesis that defective BRCA1 results in malfunctioning centrosomes and faulty centrosomes are a possible cause of cancer. Using EGFP-tagged fusion proteins and BRCA1−/− cells we show that although some BRCA1 antibodies do label centrosomes under certain fixation conditions, BRCA1 is not a centrosomal protein. Therefore, it is unlikely that a mutation in BRCA1 directly alters centrosome structure and function. BRCA1 plays an established role in DNA damage repair and in G2/M checkpoint regulation. We present evidence that multiple centrosomes can arise in any cell when G2/M checkpoint fails and entrance into mitosis occurs in the presence of DNA damage.