Virpi Rantanen

Amplification and Deletion of Topoisomerase IIα Associate with ErbB-2 Amplification and Affect Sensitivity to Topoisomerase II Inhibitor Doxorubicin in Breast Cancer

Topoisomerase IIalpha (topoIIalpha) is a key enzyme in DNA replication and a molecular target for many anti-cancer drugs called topoII inhibitors. The topoIIalpha gene is located at chromosome band 17q12-q21, close to the ErbB-2 oncogene (HER-2/neu), which is the most commonly amplified oncogene in breast cancer. Because of the physical proximity to ErbB-2, copy number aberrations may also occur in the topoIIalpha gene. These topoIIalpha gene copy number aberrations may be related to the altered chemosensitivity to topoII inhibitors that breast cancers with ErbB-2 amplification are known to have. We used fluorescence in situ hybridization to study copy number aberrations of both topoIIalpha and ErbB-2 in nine breast cancer cell lines and in 97 clinical breast tumors, which were selected for the study according to their ErbB-2 status by Southern blotting. TopoIIalpha-protein expression was studied with Western blot and sensitivity to doxorubicin (a topoII inhibitor) with a 96-well clonogenic in vitro assay. Two of the five cell lines with ErbB-2 gene amplification (SK-BR-3 and UACC-812) showed amplification of topoIIalpha. In MDA-361 cells, ErbB-2 amplification (14 copies/cell) was associated with a physical deletion of topoIIalpha (four copies of chromosome 17 centromere and two copies of topoIIalpha). The topoIIalpha amplification in UACC-812 cells was associated with 5.9-fold-increased topoIIalpha protein expression and 2.5-fold-increased sensitivity to the topoII inhibitor, doxorubicin, whereas the deletion in MDA-361 leads to decreased protein expression (45% of control) and a 2.4-fold-increased chemoresistance in vitro. Of 57 ErbB-2-amplified primary breast carcinomas, 25 (44%) showed ErbB-2-topoIIalpha coamplification and 24 (42%) showed a physical deletion of the topoIIalpha gene. No topoIIalpha copy number aberrations were found in 40 primary tumors without ErbB-2 amplification. TopoIIalpha gene amplification and deletion are common in ErbB-2-amplified breast cancer and are associated with increased or decreased sensitivity to topoII inhibitors in vitro, respectively. These findings may explain the altered chemosensitivity to topoII inhibitors reported in ErbB-2-amplified breast cancers.

Impairment of heart rate variability during paclitaxel therapy

Paclitaxel, which has been reported to be effective in treating metastatic breast carcinoma and advanced ovarian carcinoma, has been associated with cardiac side effects. Therefore, the effect of paclitaxel on cardiovascular autonomic regulation was studied.

Supra-additive effect with concurrent paclitaxel and cisplatin in vulvar squamous cell carcinoma in vitro

The effect of concurrent paclitaxel and cisplatin was tested in vitro in 5 vulvar squamous cell carcinoma (SCC) cell lines (UM‐SCV‐1A, ‐2, ‐4 and ‐7 and UT‐SCV‐3). Chemosensitivity was tested using the 96‐well plate clonogenic assay. Paclitaxel concentrations used varied between 0.4 and 1.6 nM, and cisplatin concentrations varied between 0.1 and 0.9 μg/ml. These drug concentrations are clinically achievable. Survival data were fitted to the LQ model, and the area under the curve (AUC) value was obtained with numerical integration. The type of interaction was determined by comparing the AUC ratio of the 2 drugs with the survival fraction (SF) of paclitaxel alone. With all cell lines tested the growth‐inhibitory effect of simultaneous paclitaxel and cisplatin was at least additive. The effect of the tested combination on the UM‐SCV‐1A and UT‐SCV‐3 cell lines was clearly supra‐additive with all paclitaxel concentrations tested, and the UM‐SCV‐4 and UM‐SCV‐7 cell lines exhibited a supra‐additive effect with increasing paclitaxel concentrations. The degree of supra‐additivity was dose‐dependent in the UM‐SCV‐7 cell line with increasing synergy at higher paclitaxel doses. In the current study the combination of paclitaxel and cisplatin had a clear additive or supra‐additive cytotoxic effect on the vulvar SCC cell lines, and it has been successfully used in other gynecologic malignancies; therefore concurrent paclitaxel and cisplatin also deserves further testing in clinical settings in advanced‐stage vulvar carcinoma, which has a poor prognosis.

Carboplatin-paclitaxel- and carboplatin-docetaxel-induced cytotoxic effect in epithelial ovarian carcinoma in vitro

The combination of paclitaxel and cisplatin is standard for patients with newly diagnosed epithelial ovarian carcinoma. The role of another taxane, docetaxel, currently is being studied. Due to its milder nonhematologic toxicity carboplatin increasingly is being substituted for cisplatin in taxane‐based combinations. The purpose of this study was to compare the combination of carboplatin‐paclitaxel with carboplatin‐docetaxel in ovarian carcinoma in vitro, and to assess the type of interaction, if any.

Does skin fibroblast radiosensitivity predict squamous cancer cell radiosensitivity of the same individual

Individualization of radiation doses is presumed to result in better radiotherapy outcome. Success rate in measuring radiosensitivity is probably the most limiting factor for present radiosensitivity assays to be introduced into clinical routine. To find a simpler predictive parameter, we compared the radiosensitivity of dermal fibroblasts and head and neck squamous cell carcinoma (SCC) cell lines established from the same individuals. The radiosensitivity was tested using the clonogenic 96‐well plate assay. The surviving fraction at 2.0 Gy (SF2) was determined, as well as the mean inactivation dose (AUC) of cancer cells. SF2 of SCC cell lines and skin fibroblasts were 0.25–0.44 and 0.11–0.43, respectively. AUC of SCC cells was 1.4–2.1 Gy. Dermal fibroblasts were more radiosensitive than SCC cells in 14 of 15 cases. In 1 patient (UT‐SCC‐8), cancer cells were found to be more radiosensitive than corresponding dermal fibroblasts. There was a clear tendency to a correlation between radiosensitivities of these 2 cell types, but statistical significance was reached only when the data of UT‐SCC‐8 was excluded. In our material, the intrinsic radiosensitivity of head and neck SCC cells could in most cases be predicted from the intrinsic radiosensitivity of dermal fibroblasts established from the same individual.

In vitro concurrent paclitaxel and radiation of four vulvar squamous cell carcinoma cell lines

The antitubule agent paclitaxel causes a cell cycle blockage in the most radiosensitive part of the cell cycle, the G2/M phase. The possible radiosensitizing effect of paclitaxel was tested in four vulvar (UM‐SCV‐1A, UM‐SCV‐1B, UM‐SCV‐2, and UM‐SCV‐4) squamous cell carcinoma (SCC) cell lines.

Comparative evaluation of cisplatin and carboplatin sensitivity in endometrial adenocarcinoma cell lines.

Platinum analogues are frequently used in the treatment of advanced or recurrent endometrial cancer. To study the sensitivity of endometrial cancer to cisplatin and carboplatin, we tested two long-established (RL95-2, KLE) and six new cell lines (UM-EC-1, UM-EC-2, UM-EC-3, UT-EC-2A, UT-EC-2B, UT-EC-3) using the 96-well-plate clonogenic assay. This assay has proven to be suitable for testing chemosensitivity of both adenocarcinoma and squamous cell carcinoma. The chemosensitivity was expressed as an IC50 value, the drug concentration causing 50% inhibition of clonogenic survival. IC50 values were obtained from dose-response curves after fitting the data by the linear quadratic equation, F = exp[-(alpha D + beta D2)]. The IC50 values of the two platinum derivatives varied considerably. The values for cisplatin varied between 0.022 microgram ml-1 and 0.56 microgram ml-1 and the corresponding values for carboplatin were 0.096-1.20 microgram ml-1. The range of the ratios between carboplatin IC50 and cisplatin IC50, from 1.5:1 to 4.4:1, was rather narrow. However, no constant ratio between carboplatin IC50 and cisplatin IC50 could be detected. The equivalent doses with regard to efficacy of these two platinum analogues remain to be determined.

Paclitaxel combined with fractionated radiation in vitro: A study with vulvar squamous cell carcinoma cell lines

Concurrent paclitaxel and radiation has given promising results in the treatment of a variety of solid tumors. We wanted to test the efficacy of this combination for vulvar carcinoma, which currently has a poor outcome in advanced stages. The radiation sensitivity, sublethal damage repair (SLDR) capacity and effect of paclitaxel during fractionated radiation were assessed in our study on 7 vulvar inherently radioresistant squamous cell carcinoma (SCC) cell lines. The 96‐well plate clonogenic assay was used. Survival data were fitted to the linear quadratic model. The area under the curve (AUC), equivalent to mean inactivation dose (D̄), was obtained with numerical integration. AUC ratios between single‐dose radiation and fractionated radiation with or without paclitaxel were used to determine the SLDR of the cell lines and the effect of paclitaxel on it. Seven currently tested vulvar SCC cell lines were found to have a limited capacity of repairing sublethal damage (SLD). Only 3 of them presented SLDR of significance. The effect of concurrent radiation and paclitaxel was clearly additive when the radiation dose was fractionated in most of the cell lines. In addition, 2 of the cell lines having SLDR exhibited a trend toward losing the repair capacity when paclitaxel was present during the irradiation. In addition, the survival curve of the UM‐SCV‐1A cell line gave the impression of a true paclitaxel effect on SLDR. Paclitaxel used concurrently with fractionated radiation showed effectiveness on vulvar carcinoma. The effect was at least additive and could even be expected to abrogate the SLDR during split‐dose radiation.

Paclitaxel changes sympathetic control of blood pressure

Paclitaxel has become part of standard therapy in the treatment of ovarian and breast cancer. Concern has been raised about the effects of paclitaxel on cardiovascular function. Therefore, this study of the effects of paclitaxel on autonomic cardiovascular control was initiated. Eighteen women treated for ovarian or breast cancer were examined with autonomic cardiovascular function tests, once before the treatment and once after the second course of paclitaxel. Heart rate and blood pressure variability and changes in heart rate and blood pressure responses to the tests were measured. Baroreflex sensitivity was calculated from the Valsalva manoeuvre non-invasively. Paclitaxel did not change heart rate variability at rest compared with the pretreatment level. However, medium frequency variability of blood pressure was smaller after treatment with paclitaxel. Paclitaxel treatment did not impair the heart rate and blood pressure responses to the autonomic function tests. The results do imply that paclitaxel alters sympathetic control of blood pressure. Nevertheless, paclitaxel does not appear to precipitate autonomic cardiac neuropathy.

Radiation sensitivity of endometrial carcinoma in vitro

The role of radiation therapy is essential in the treatment of endometrial carcinoma. The present knowledge of the radiation sensitivity of endometrial carcinoma is mostly empirical and based on clinical trials. To study the inherent radiation sensitivity of endometrial carcinoma, we tested two long-established cell lines (RL95-2, KLE) and four new, low-passage cell lines (UM-EC-1, UM-EC-2, UM-EC-3, UT-EC-1) by using a 96-well plate clonogenic assay. This method has proved to be suitable for clonogenic studies of both squamous cell carcinomas (SCC) and adenocarcinomas in our recent works. Plating efficiencies of the cell lines tested varied from 0.005 to 0.45. Cells were irradiated in suspension with a 4-MeV linear accelerator at a dose rate of 2.0 Gy/min. Survival data were fitted by the linear quadratic function F = exp (-(alpha D + beta D2)). Radiation sensitivity was expressed as the area under the curve (AUC), equivalent to the mean inactivation dose. UM-EC-1 and UT-EC-1 were the most radiation-resistant cell lines tested (AUC greater than 1.6 Gy), while UM-EC-3 was the most sensitive (AUC = 1.0 Gy). The difference in radiation sensitivity between these cell lines was statistically significant (P less than 0.001). As a group, endometrial carcinoma cell lines were clearly more radiosensitive than most SCC lines of head and neck and vulva earlier tested by us. However, our results showed also great variance in the inherent radiation sensitivity between individual cell lines derived from endometrial carcinomas.