Martina Becker

Cisplatin, doxorubicin and paclitaxel induce mdr1 gene transcription in ovarian cancer cell lines

The clinical observation of the multidrug resistance (MDR) phenotype is often associated with overexpression of the mdrl gene, in particular with respect to ovarian cancer. However, until now the mdrl-inducing potential of commonly used antineoplastics has been only incompletely explored. We performed short-term cultures of six ovarian cancer cell lines (MZOV4, EF027, SKOV3, OAW42, OTN14, MZOV20) exposed to either blank medium or cisplatin, doxorubicin or paclitaxel at concentrations related to the clinically achievable plasma peak concentration. A highly specific quantitative real-time RT-PCR was used to detect the Mdr1 transcripts. Mdrl mRNA contents were calibrated in relation to coamplified GAPDH mRNA. Mdrl mRNA was detectable in each cell line. In 13 out of 18 assays (72%) the specific anticancer drug being tested induced mdr1 transcription. No decrease in mdr1 mRNA concentration was observed. Our data suggest that mdr1 induction by antineoplastics is one of the reasons for failure of ovarian cancer therapy but may vary individually.

Association of the vitamin D receptor genotype with bone metastases in breast cancer patients.

This study was designed in order to evaluate specific vitamin D receptor (VDR) genotypes as indicators of the likelihood of developing osseous metastases in breast cancer patients. Therefore, we determined polymorphisms of the VDR gene in a study group comprising 183 breast cancer patients. Specific fragments spanning over intron 8 and exon 9 of the VDR gene were amplified by polymerase chain reaction. The fragments were then incubated with each of the specific endonucleases ApaI, BsmI or TaqI, respectively. The VDR gene polymorphisms were detected by the presence or absence of the particular restriction site using agarose gel electrophoresis. Statistical analyses revealed a significant correlation between both the VDR gene polymorphisms indicated as AA (absence of the ApaI restriction site in both alleles) or TT (absence of the TaqI restriction site in both alleles), respectively, and the occurrence of bone metastases. Patients with the AA genotype have a 1.7-fold increased risk of developing bone metastases, whereas patients with the TT genotype have a 0.5-fold risk. Neither other genotypes nor allelic combinations displayed any further correlation with the clinical stage. The data suggest that the AA genotype of the VDR gene might be useful to identify breast cancer patients with a high probability of forming occult bone metastases who are considered to benefit from an adjuvant bone-protective therapy.

The V109G Polymorphism of the p27 Gene CDKN1B Indicates a Worse Outcome in Node-Negative Breast Cancer Patients

Although p27 plays a central role in cell cycle regulation, its role in breast cancer prognosis is controversial. Furthermore, the p27 gene CDKN1B carries a polymorphism with unknown functional relevance. This study was designed to evaluate p27 expression and p27 genotyping with respect to early breast cancer prognosis. 279 patients with infiltrating metastasis-free breast cancer were included in this study. p27 expression was determined in tumor tissue specimens from 261 patients by immunohistochemistry. From 108 patients, the CDKN1B genotype was examined by PCR and subsequent direct sequencing. 55.2% of the tumors were considered p27 positive. p27 expression did not correlate with any of the established parameters except for nodal involvement but significantly correlated to prolonged disease-free survival. In 35% of the tumors analyzed, the CDKN1B gene showed a polymorphism at codon 109 (V109G). The V109G polymorphism correlated with greater nodal involvement. In the node-negative subgroup, V109G correlated significantly with a shortened disease-free survival. In conclusion, the determination of the CDKN1B genotype might be a powerful tool for the prognosis of patients with early breast cancer.

Time to progression is dependent on the expression of the tumour suppressor PTEN in ovarian cancer patients

Background Quantitative analyses of PTEN expression of ovarian cancer tissues were performed in this study. PTEN expression was investigated in terms of each patients progression‐free interval to indicate the role of PTEN in the generation of platinum refractory tumours.

Immunohistochemical detection of H-ras protooncoprotein p21 indicates favorable prognosis in node-negative breast cancer patients.

We tested primary breast carcinoma tissues from 297 patients using a monoclonal antibody (clone 235-1.7.1.) for the expression of p21ras. 58% of tumors were p21ras-positive. When calculated in a univariate fashion, p21 expression correlated with proliferation activity (proliferating cell nuclear antigen) only. Using the log-rank test (median observation time 94 months) a significantly worse prognosis (disease-free survival, overall survival) relation was found with larger tumors, nodal involvement, anaplasia, rising proliferation activity, and lack of steroid receptors. Detection of p21ras correlated with a more favorable prognosis but only in node-negative patients. Stepwise correlation according to the Cox hazard model ranked p21 expression as a most significant predictor of prognosis second only to nodal status. These data suggest that detection of p21ras indicates the presence of a parameter which may act as tumor suppressor and benefit patients’ survival.

Amplification of the mdr1-gene is uncommon in recurrent ovarian carcinomas

Ovarian carcinomas are known to rapidly develop drug resistance against chemotherapeutic agents. This phenomenon is often associated with the expression of pl70-glycoprotein. A high rate of transcription of the corresponding mdr1-gene in resistant tumors is reported. Amplification of the mdr1-gene has been observed in tumor cell lines exposed to cytotoxic drugs; however, significant information is lacking as to whether this holds true in clinical carcinomas. To fill this gap, we investigated the rate of gene amplification of the mdr1-gene in 63 recurrent ovarian carcinomas and we determined the resistance pattern of these cells using an ex vivo assay. The tumors showed varying ex vivo resistance patterns which did not correlate to clinical parameters. Amplification of the mdr1-gene was not observed in any of the cancer specimens. Therefore, we conclude that mdr1-gene amplification is not a common pathway for the development of chemoresistance in clinical ovarian carcinomas.

High Apoptotic Index Correlates to p21 and p27 Expression Indicating a Favorable Outcome of Primary Breast Cancer Patients, but Lacking Prognostic Significance in Multivariate Analysis

This study was performed in order to investigate the role of the apoptotic index (AI) as a prediction parameter for the prognosis of patients with primary breast cancer. AI was determined by DNA fragmentation on 298 primary breast cancer samples and compared to clinically established breast cancer parameters. Additionally, we determined the expression of functional parameters including proliferating cell nuclear antigen, p21waf and p27kip by immunohistochemistry. The mean AI was found to be 11.9% (range, 0–90%). 189 tumors (63.4%) were negative for apoptosis, while 109 tissue samples (36.6%) were apoptotic with >5% positive cells. Using univariate analysis (χ2 test), the AI did not show any significant correlation to one of the established prognostic parameters of primary breast cancer (p > 0.05). In contrast, we found a significant positive correlation to the expression of the cell cycle inhibitors p21waf (p = 0.04) and p27kip (p = 0.024). During the clinical follow-up (median observation time for disease-free survival 87 months), several clinically established prognostic parameters including menopausal status, nodal status, tumor size, tumor grade, and hormone receptor expression could be confirmed and were analyzed with respect to the AI in the tumor. Furthermore, AI displayed a significant positive correlation to disease-free survival using Kaplan-Meier survival analysis (log-rank test, p = 0.04). However, AI lost its prognostic significance in multivariate analysis based on the Cox proportional hazard model (relative risk 0.8, confidence interval 0.52–1.33, p = 0.44). Our data indicate that high apoptotic rates in cancer tissues are indicative of a favorable patient outcome. However, the AI was not an independent factor. The study provides indirect evidence that this process may involve cell cycle inhibitors physiologically.

Interaction of cisplatin, paclitaxel and adriamycin with the tumor suppressor PTEN.

Due to its pivotal role in signal transduction, the universal tumor suppressor PTEN (also termed MMAC or TEP) is one of the putative candidates for involvement in tumorigenesis of several tissues. Although involvement of PTEN in tumorigenesis was shown in different tissues, no data are available concerning PTEN activity in response to antineoplastic agents. Therefore, we assayed the PTEN activity exposed to either blank medium or the commonly used anti-cancer drugs cisplatin, adriamycin or paclitaxel, respectively, in three different concentrations. PTEN activity was determined using the Malachite Green assay basing upon dephosphorylation of phosphatidylinositol-3,4,5-triphosphate (PIP3) by the PTEN enzyme and subsequent determination of inorganic phosphate released. Although the three different anti-cancer drugs assayed act with different cellular modes, the antineoplastics influenced PTEN activity in a similar manner: at low concentrations tested all three antineoplastics significantly increased PTEN activity. However, increasing drug concentrations exhibited a decline but not a total loss of PTEN activity. The data indicate that PTEN activity is increased following cytotoxic drug exposure and, thereby, exhibits its suppressive function. However, the decrease of PTEN activity in response to increasing drug concentrations suggests an aberration of total functional activity. As far as the regulative checkpoint PTEN is abolished, tumor cells might evade cell death pathways resulting in increased proliferation of cancer cells. This might be a general event in refractory tumor cells surviving chemotherapy.

Heterogeneity of proteinkinase C activity and PKC-ζ expression in clinical breast carcinomas

Abstract Proteinkinase C (PKC) is involved in carcinogenesis, proliferation, and metastatic spread of breast cancer. New anticancer strategies have been developed with PKC as a potential target for therapeutic intervention. However, most of the encouraging preliminary data were observed in breast cancer cell lines only. Insignificant information is available concerning clinical breast cancer cells. Our aim was to investigate the involvement of PKC in clinical breast carcinoma cells. To this end, we set up short-term cultures (3 days) of native tumor cells derived from 12 patients with advanced breast cancer. Addition of commonly used antineoplastics, including both single agents and combinations (tamoxifen, Adriamycin, paclitaxel, Adriamycin plus paclitaxel, epirubicin plus 4-OOH-cyclophosphamide, mitoxantrone, mitoxantrone plus vinorelbin, vinorelbin), simulated the clinical situation. In relation to each control we determined total PKC activity and quantified the PKC-ζ isoform. In 6 patients, no obvious alteration of PKC activities was detected. In the remainder, either inhibition or augmentation of PKC activity in the presence of cytostatics was detected. However, no tendency could be observed concerning the influence of the therapeutics on PKC activity. PKC-ζ expression was much more heterogeneous than activity assays. Although anticancer drugs influenced PKC-ζ expression, the results showed no uniformity with regard to PKC-ζ expression. Moreover, PKC-ζ expression did not correlate with total PKC activity, indicating a differential expression of different PKC isoenzymes. Therefore, we conclude that both PKC activity and PKC-ζ expression differ individually. More data concerning this topic are necessary prior to offering a clinically useful PKC-tailored regimen.

Dysregulation of protein kinase C activity in chemoresistant metastatic breast cancer cells

This study was performed to evaluate the role of protein kinase C (PKC) activity in the development of chemoresistance in clinical breast cancer cells. To simulate the clinical situation, native tumor cells derived from 10 patients with advanced breast cancer were brought into short-term cultures, and treated with anthracyclines (doxorubicin, mitoxantrone), paclitaxel and combinations, respectively. After 3 days of incubation, we determined total PKC activity relative to each control incubated with blank medium. Furthermore, we determined the chemoresistance against these drugs from each cell population separately. Relative PKC activity ranged from 14 to 249%; 64% (37 of 58) of the breast cancer cell suspensions were considered chemoresistant. There was a non-significant trend to a higher relative PKC activity in resistant cells compared to non-resistant cells (p=0.058), regardless of the antineoplastic agent investigated. The individual variability in both PKC activity and chemoresistance pattern revealed that dysregulated PKC activity mediates resistance to antineoplastics. In order to achieve clinical value, evaluation of more data concerning the PKC signal-transduction pathway is necessary. New protocols of cancer treatment will require this information in order to be successful.