Checkpoint kinase 2 (Chk2) supports sensitivity to platinum-based treatment in high grade serous ovarian cancer.

OBJECTIVE: Platinum-based chemotherapy is the standard treatment in advanced stage high grade serous ovarian cancer (HGSOC), but the majority of patients will relapse with drug-resistant disease. Platinum induces double-strand DNA breaks and subsequently activation of the DNA damage response (DDR). Drugs targeting DDR pathway components have gained major interest to be combined with chemotherapy as they could increase the therapeutic window. In the present study, we investigated the activation status of the Ataxia Telangiectasia Mutated (ATM) signaling axis within the DDR in a large, well-defined cohort of advanced stage HGSOC patients. METHODS: Pre-therapy activation status of the ATM signaling axis of the DDR was determined by immunohistochemistry in 125 chemo-naive advanced stage HGSOC patients. Ovarian cancer cell lines with stable checkpoint kinase 2 (Chk2) knock down were used to study cell cycle distribution and survival in long-term clonogenic survival assays. RESULTS: All ATM signaling axis components showed high expression levels. In two well-defined groups with the largest contrast in treatment response, high expression of Chk2 was related to good response (OR=0.132; P=0.014). Chk2 depletion abrogated the cisplatin-induced S-phase cell cycle arrest and caused increased resistance to cisplatin in long-term clonogenic survival assays. CONCLUSIONS: Chk2 is related to good response to platinum-based chemotherapy in advanced stage HGSOC patients. Chk2-depleted ovarian cancer cell lines have diminished platinum sensitivity, suggesting that Chk2 should not be considered a therapeutic target along with platinum-based treatment in HGSOC patients.

Drug-induced caspase 8 upregulation sensitises cisplatin-resistant ovarian carcinoma cells to rhTRAIL-induced apoptosis.

BACKGROUND: Drug resistance is a major problem in ovarian cancer. Triggering apoptosis using death ligands such as tumour necrosis factor-related apoptosis inducing ligand (TRAIL) might overcome chemoresistance. METHODS: We investigated whether acquired cisplatin resistance affects sensitivity to recombinant human (rh) TRAIL alone or in combination with cisplatin in an ovarian cancer cell line model consisting of A2780 and its cisplatin-resistant subline CP70. RESULTS: Combining cisplatin and rhTRAIL strongly enhanced apoptosis in both cell lines. CP70 expressed less caspase 8 protein, whereas mRNA levels were similar compared with A2780. Pre-exposure of particularly CP70 to cisplatin resulted in strongly elevated caspase 8 protein and mRNA levels. Caspase 8 mRNA turnover and protein stability in the presence or absence of cisplatin did not differ between both cell lines. Cisplatin-induced caspase 8 protein levels were essential for the rhTRAIL-sensitising effect as demonstrated using caspase 8 small-interfering RNA (siRNA) and caspase-8 overexpressing constructs. Cellular FLICE-inhibitory protein (c-FLIP) and p53 siRNA experiments showed that neither an altered caspase 8/c-FLIP ratio nor a p53-dependent increase in DR5 membrane expression following cisplatin were involved in rhTRAIL sensitisation. CONCLUSION: Cisplatin enhances rhTRAIL-induced apoptosis in cisplatin-resistant ovarian cancer cells, and induction of caspase 8 protein expression is the key factor of rhTRAIL sensitisation.

Abundant Fas expression by gastrointestinal stromal tumours may serve as a therapeutic target for MegaFasL.

Although the tyrosine kinase inhibitor imatinib has been shown to be an active agent in patients with gastrointestinal stromal tumours (GIST), complete remissions are almost never seen and most patients finally experience disease progression during their course of treatment. An alternative therapeutic option is to target death receptors such as Fas. We showed that a panel of imatinib-sensitive (GIST882) and imatinib-resistant (GIST48, GIST430 and GIST430K-) cell lines expressed Fas. MegaFasL, a recently developed hexameric form of soluble Fas ligand (FasL), appeared to be an active apoptosis-inducing agent in these cell lines. Moreover, MegaFasL potentiated the apoptotic effects of imatinib. Immunohistochemical evaluations, in 45 primary GISTs, underscored the relevance of the Fas pathway: Fas was expressed in all GISTs and was expressed strongly in 93%, whereas FasL was expressed at moderate and strong levels in 35 and 53% of GISTs, respectively. Fas and FasL expression were positively correlated in these primary GISTs, but there was no association between Fas or FasL expression and primary site, histological subtype, tumour size, mitotic index, risk classification, and KIT mutation status. The abundant immunohistochemical Fas and FasL expression were corroborated by western blot analysis. In conclusion, our data implicate Fas as a potential therapeutic target in GIST.

Relationship of cellular glutathione to the cytotoxicity and resistance of seven platinum compounds.

The role of glutathione (GSH) in the effectiveness of and resistance to 7 platinum compounds [5 Pt(II) and 2 Pt(IV) drugs] was evaluated in a 8.6-fold cisplatin (CDDP)-resistant human small cell lung cancer cell line (GLC4/CDDP), the parent GLC4 line, a 3.7-fold CDDP-resistant human embryonal carcinoma cell line (Tera-CP), and the parent Tera line (NTera2/D1). Resistance factors for both CDDP-resistant cell lines were determined after continuous incubation (4 days) with CDDP. Continuous incubation with the other studied platinum drugs revealed complete cross-resistance for carboplatin (CBDCA) and zeniplatin but less for enloplatin (ENLO) and iproplatin in both models. Tetraplatin and lobaplatin showed, respectively, partial and complete cross-resistance in GLC4/CDDP but no cross-resistance in Tera-CP. GSH level, but not glutathione S-transferase activity, of the 4 cell lines correlated with platinum drug concentrations inhibiting cell survival by 50% after continuous incubation (r = 0.86, P < 0.05). GSH depletion by DL-buthionine-S,R-sulfoximine (BSO) increased sensitivity, as measured after a 4-h exposure to the drugs, of GLC4/CDDP for CDDP 2.0-fold, for CBDCA 1.7-fold, for zeniplatin 1.7-fold, and almost to the level of the sensitive GLC4 for ENLO, whereas no effect was observed for lobaplatin and the Pt(IV) compounds iproplatin and tetraplatin. BSO-modulating effect was higher in the sensitive GLC4 line for most compounds; therefore reduction of resistance could be achieved only for CDDP and ENLO. In contrast to GLC4, no modulation occurred in Tera. In Tera-CP BSO increased sensitivity for CDDP 1.5-fold, for CBDCA 1.9-fold, and for zeniplatin 1.2-fold; no effect was observed for ENLO, lobaplatin, and the Pt(IV) compounds. Reduction of CDDP resistance by BSO was known to occur with identical cellular platinum levels and higher Pt-DNA binding in GLC4/CDDP. However, pretreatment with BSO followed by 4 h ENLO incubation increased cellular platinum levels in both GLC4 and GLC4/CDDP while Pt-DNA binding remained unchanged. In conclusion, GSH reflected sensitivity to platinum-containing drugs. However, since the involvement of GSH differed between the models and the various platinum drugs, the effect of modulation with BSO was unpredictable.

Effects of amiodarone, cyclosporin A, and PSC 833 on the cytotoxicity of mitoxantrone, doxorubicin, and vincristine in non-P-glycoprotein human small cell lung cancer cell lines.

The multidrug resistance (MDR) modulators amiodarone (AM), cyclosporin A (CsA), and PSC 833 were tested for their potential to modulate cytotoxicity of doxorubicin (DOX), vincristine (VCR), and mitoxantrone (MX) in a sensitive human small cell lung carcinoma cell line GLC4, in its DOX-resistant non-P-glycoprotein subline GLC4-Adr, and in its cisplatin-resistant subline GLC4-CDDP. GLC4-Adr, in which overexpression of the so-called multidrug resistance-associated protein has been demonstrated, is 91-fold resistant for DOX, 22-fold for VCR, and 7.5-fold for MX, compared with its sensitive cell line. AM previously modulated DOX and VCR resistance in the P-glycoprotein-positive human colon cancer cell line COLO 320. Cytotoxicity was studied in the microtiter well tetrazolium assay. In the small cell lung carcinoma cell lines described above, AM did not increase cytotoxicity of DOX, but increased VCR cytotoxicity; moreover, AM was shown to be a potent modulator of MX cytotoxicity. CsA did not potentiate DOX cytotoxicity, but, at a concentration of 4 microM, it modestly increased VCR cytotoxicity in GLC4. However, 0.8 and 4.0 microM CsA protected against MX cytotoxicity in GLC4 and GLC4-CDDP, but no effect was observed in GLC4-Adr. At the much higher ID10 concentration CsA modulated MX cytotoxicity 1.6-fold in GLC4-Adr and slightly in GLC4 and GLC4-CDDP. PSC 833, a nonimmunosuppressive CsA analogue, did not alter the cytotoxicity of DOX or MX in these cell lines, but potentiated VCR cytotoxicity in GLC4-Adr at a concentration of 0.4 microM. The modulation of MX cytotoxicity by AM and the protection by CsA was confirmed in a clonogenic assay. In the colony-forming unit granulocyte-monocyte assay, no additional MX toxicity on normal bone marrow by AM was observed. Flow cytometry of cellular MX fluorescence was performed in order to elucidate the mechanism behind the AM-induced increased MX cytotoxicity. This revealed an increase in cellular MX after 1-h incubation of MX combined with AM and an inhibition of efflux from GLC4 and GLC4-Adr; CsA and PSC 833 had no effect on MX efflux. An increase in MX-induced cleavable complexes by AM in GLC4 was observed using the K+/sodium dodecyl sulfate coprecipitation assay, but no effect of CsA was found. In conclusion, AM enhances MX and VCR cytotoxicity in these sensitive, non-P-glycoprotein DOX and cisplatin-resistant small cell lung carcinoma cell lines. It also inhibits efflux of MX and causes more MX-induced cleavable complexes.

Antagonism of HSV-tk transfection and ganciclovir treatment on chemotherapeutic drug sensitivity.

Our study focused on the influence of herpes simplex virus thymidine kinase (HSV-tk) expression and ganciclovir (GCV) treatment on the sensitivity of C6 glioma cells to frequently used chemotherapeutic drugs, i.e. adriamycin (ADR), cisplatin (CDDP), 5-fluorouracil (5-FU), and methotrexate (MTX). Transfection with HSV-tk revealed an increased sensitivity to GCV and CDDP and a decreased sensitivity to ADR and MTX. No significant differences were found in sensitivity to 5-FU. Combined treatment in a HSV-tk negative cell line revealed an additive effect when GCV was combined with ADR, whereas an antagonistic effect was found when GCV was combined with CDDP, 5-FU, or MTX. Comparable results were obtained in an HSV-tk positive cell line, apart from CDDP, which showed an additive effect. In conclusion, both HSV-tk transfection and subsequent GCV treatment can influence the sensitivity of tumor cells to various chemotherapeutic drugs in an antagonistic manner. Therefore, combining HSV-tk/GCV gene therapy with chemotherapy might not always be beneficial.

Telomerase in (pre)neoplastic cervical disease.

This study was performed to determine upregulation of the human telomerase RNA component (hTR) and mRNA of the catalytic subunit of telomerase (hTERT) in (pre)malignant cervical lesions, to analyze possible intralesional heterogeneity of hTR expression, and to relate hTR and hTERT mRNA levels to telomerase activity levels and human papillomavirus (HPV) typing. hTR expression was determined by in situ hybridization (ISH) on paraffin-embedded sections, obtained from patients with cervical intraepithelial neoplasia (CIN) I-III or cervical cancer and from normal controls. hTR and hTERT mRNA expression were determined by semiquantitative rt-PCR on frozen samples from the same lesions. Data on telomerase activity and HPV were obtained from a previous study. hTR expression as determined by ISH was observed in 0 of 8 normal cervices, 1 of 14 CIN I, 15 of 28 CIN II, 21 of 30 CIN III, and 16 of 18 cervical cancer specimens. In general, hybridization patterns for hTR expression were homogeneous throughout the lesion. Frequency of hTR expression was related to grade of CIN/cervical cancer (P<.001). hTR expression, as determined by rt-PCR, was detected in 8 of 8 normal cervices, 2 of 2 CIN I, 12 of 14 CIN II, 23 of 23 CIN III, and 16 of 17 cervical cancer specimens. hTERT mRNA was detected in 1 of 8 normal cervices, 1 of 2 CIN I, 5 of 14 CIN II, 14 of 23 CIN III, and 11 of 17 cervical cancer specimens. hTR as determined by rt-PCR was not related to grade of CIN/cervical cancer, whereas hTERT mRNA expression was related to grade of CIN/cervical cancer (P<.01). hTR expression, as determined by ISH and hTERT mRNA expression by rt-PCR, were related to telomerase activity levels (P<.001, P<.05, respectively) and presence of oncogenic types of HPV (both P<.05). Our data show frequent upregulation of hTR and hTERT mRNA expression in CIN lesions, which appear to occur earlier than induction of telomerase activity. The fact that semiquantitative hTERT mRNA as well as hTR levels are related to telomerase activity levels illustrates that in (pre)malignant cervical lesions upregulation of both telomerase components may be important for functional telomerase.

Thymidylate synthase levels in tumor biopsies from patients with colorectal cancer.

Catalytic activity and FdUMP binding characteristics of thymidylate synthase (TS) were determined in 22 tumor biopsies of patients to be treated (15) or just treated (7) for colorectal cancer with 5-fluorouracil and leucovorin. In 19 samples both parameters could be determined and were found to represent a wide range (15-20 fold). High values of activity correlated with no response, but low values of binding or activity were found in responders as well as in non-responders. It is concluded that the determination of parameters related to TS function is feasible in tumor biopsies and its relevance for clinical management deserves further study.

A PCR-aided transcript titration assay (PATTY) to measure topoisomerase I gene expression in human tumor specimens.

UNLABELLED: Topoisomerase I (topo I) inhibitors are promising anticancer agents with demonstrated activity against a wide range of solid tumors. Quantitative information on topol mRNA levels in tumor biopsies may predict response to topo I inhibitors. MATERIALS AND METHODS: A polymerase chain reaction aided transcript titration assay (PATTY) was developed to allow quantitation of topol mRNA in small samples. Concentrations of topol mRNA in total RNA samples were estimated by RT-PCR analysis in a human small cell lung cancer (SCLC) cell line (GLC,) and its topotecan (GL2C/SK and F) and camptothecin (GL2C/Campt) resistant sublines, human non-small cell lung cancer (NSCLC) and ovarian carcinoma samples. RESULTS: Topol PATTY showed a decreased topo I mRNA level in GLC2/SK and F (4.5 pg/100 ng total RNA) and GLC,/Campt (2.2 pg/100 ng total RNA), respectively, compared to the parent cell line GLC2 (5.4 pg/100 ng total RNA). Topol protein levels as measured by Western blotting were compatible with topol mRNA levels. Median (range) topol mRNA levels were 3.23 (2.33-5.10) pg/100 ng total RNA in resected NSCLC specimen (n = 6), and 2.03 (0.54-0.95) pg/100 ng total RNA in resected ovarian cancer specimen (n = 6). CONCLUSION: We conclude that topol PATTY is a new assay that quantitates topol mRNA levels in cell lines and small tumor samples.

A quantitative reverse transcriptase polymerase chain reaction-based assay to detect carcinoma cells in peripheral blood.

The presence of tumour cells in the circulation may predict disease recurrence and metastasis. To improve on existing methods of cytological or immunocytological detection, we have developed a sensitive and quantitative technique for the detection of carcinoma cells in blood, using the reverse transcriptase polymerase chain reaction (RT-PCR) identifying transcripts of the pancarcinoma-associated tumour marker EGP-2 (KSA or 17-1A antigen). The amount of EGP2 mRNA was quantified using an internal recombinant competitor RNA standard with known concentration and which is both reversely transcribed and co-amplified in the same reaction, allowing for a reliable assessment of the initial amount of EGP2 mRNA in the sample. Calibration studies, seeding blood with MCF-7 breast carcinoma cells, showed that the assay can detect ten tumour cells among 1.0 x 10(6) leucocytes. The PCR assay revealed that normal bone marrow expresses low levels of EGP2 mRNA, although immunocytochemistry with the anti-EGP2 MAb MOC31 could not identify any positively stained cell. Analyses using this RT-PCR assay may prove to have applications to the assessment of circulating tumour cells in clinical samples.

Quantitation of DNA topoisomerase II alpha messenger ribonucleic acid levels in a small cell lung cancer cell line and two drug resistant sublines using a polymerase chain reaction-aided transcript titration assay.

BACKGROUND: We have modified a polymerase chain reaction (PCR)-aided transcript titration assay (1) in order to allow quantitation of low amounts of DNA topoisomerase II alpha mRNA in small RNA samples. EXPERIMENTAL DESIGN: The titration assay was used to quantitate the amount of DNA topoisomerase II alpha mRNA in a human small cell lung carcinoma cell line, GLC4 and its drug-resistant sublines, GLC4/ADR and GLC4/CDDP. These cell lines show differences in DNA topoisomerase II alpha protein level and DNA topoisomerase II enzyme activity. To validate the titration assay, the results were compared with the results of a DNA topoisomerase II enzyme activity assay and DNA topoisomerase II alpha northern and western blotting assays. RESULTS: Using the titration assay, we were able to quantitate DNA topoisomerase II alpha mRNA on a picogram level starting with less than 1 micrograms of total RNA/cell line. GLC4/ADR showed a markedly decreased DNA topoisomerase II alpha mRNA level that seemed to be unchanged in GLC4/CDDP when compared with the parental cell line. The results obtained with this assay are confirmed by the western blot data and are not in contradiction with the northern blot results obtained for the three cell lines. CONCLUSIONS: The DNA topoisomerase II alpha titration assay is a highly sensitive new technique to study the role of DNA topoisomerase II alpha in drug resistance and may help to identify cancer types and patients most likely to respond to DNA topoisomerase II targeted drugs. The decrease in DNA topoisomerase II alpha protein level and DNA topoisomerase II activity in GLC4/ADR may result from transcriptional down regulation of DNA topoisomerase II alpha.