Mismatch repair and treatment resistance in ovarian cancer

BACKGROUND: The treatment of ovarian cancer is hindered by intrinsic or acquired resistance to platinum-based chemotherapy. The aim of this study is to determine the frequency of mismatch repair (MMR) inactivation in ovarian cancer and its association with resistance to platinum-based chemotherapy. METHODS: We determined, microsatellite instability (MSI) as a marker for MMR inactivation (analysis of BAT25 and BAT26), MLH1 promoter methylation status (methylation specific PCR on bisulfite treated DNA) and mRNA expression of MLH1, MSH2, MSH3, MSH6 and PMS2 (quantitative RT-PCR) in 75 ovarian carcinomas and eight ovarian cancer cell lines RESULTS: MSI was detected in three of the eight cell lines i.e. A2780 (no MLH1 mRNA expression due to promoter methylation), SKOV3 (no MLH1 mRNA expression) and 2774 (no altered expression of MMR genes). Overall, there was no association between cisplatin response and MMR status in these eight cell lines. Seven of the 75 ovarian carcinomas showed MLH1 promoter methylation, however, none of these showed MSI. Forty-six of these patients received platinum-based chemotherapy (11 non-responders, 34 responders, one unknown response). The resistance seen in the eleven non-responders was not related to MSI and therefore also not to MMR inactivation. CONCLUSION: No MMR inactivation was detected in 75 ovarian carcinoma specimens and no association was seen between MMR inactivation and resistance in the ovarian cancer cell lines as well as the ovarian carcinomas. In the discussion, the results were compared to that of twenty similar studies in the literature including in total 1315 ovarian cancer patients. Although no association between response and MMR status was seen in the primary tumor the possible role of MMR inactivation in acquired resistance deserves further investigation

MicroRNA markers for forensic body fluid identification obtained from microarray screening and quantitative RT-PCR confirmation

MicroRNAs (miRNAs) are non-protein coding molecules with important regulatory functions; many have tissue-specific expression patterns. Their very small size in principle makes them less prone to degradation processes, unlike messenger RNAs (mRNAs), which were previously proposed as molecular tools for forensic body fluid identification. To identify suitable miRNA markers for forensic body fluid identification, we first screened total RNA samples derived from saliva, semen, vaginal secretion, and venous and menstrual blood for the expression of 718 human miRNAs using a microarray platform. All body fluids could be easily distinguished from each other on the basis of complete array-based miRNA expression profiles. Results from quantitative reverse transcription PCR (RT-PCR; TaqMan) assays for microarray candidate markers confirmed strong over-expression in the targeting body fluid of several miRNAs for venous blood and several others for semen. However, no candidate markers from array experiments for other body fluids such as saliva, vaginal secretion, or menstrual blood could be confirmed by RT-PCR. Time-wise degradation of venous blood and semen stains for at least 1 year under lab conditions did not significantly affect the detection sensitivity of the identified miRNA markers. The detection limit of the TaqMan assays tested for selected venous blood and semen miRNA markers required only subpicogram amounts of total RNA per single RT-PCR test, which is considerably less than usually needed for reliable mRNA RT-PCR detection. We therefore propose the application of several stable miRNA markers for the forensic identification of blood stains and several others for semen stain identification, using commercially available TaqMan assays. Additional work remains necessary in search for suitable miRNA markers for other forensically relevant body fluids

Expression level of TOP2A in human neurofibroma and MPNST samples and cell lines.

<p>(<b>A</b>) qRT-PCR was used to determine mRNA levels of <i>TOP2A</i> in paired plexiform neurofibroma (NF, blue, n = 9) and MPNST (red, n = 9) formalin-fixed paraffin-embedded tumor samples, each pair being derived from the same NF1 patient. Asterisk indicates P<0.05. (<b>B</b>) qRT-PCR was used to determine mRNA levels of <i>TOP2A</i> in fresh frozen MPNST (red, n = 11), plexiform neurofibroma (blue, n = 7) and atypical neurofibroma (grey, n = 4). (<b>C</b>) qRT-PCR was used to determine mRNA levels of <i>TOP2A</i> in a cell line panel: Hs53.T neurofibroma cell line (blue) and STS26T, sNF96.2, ST88-14, T265 and 90-8TL MPNST cell lines (red). (<b>D</b>) Western blot displaying TOP2A protein expression in cell line panel and HEK293T. ẞ-actin levels are shown as a loading control.</p

Patient and tumor characteristics.

<p>Patient and tumor characteristics.</p

siRNA mediated knockdown of EZH2 and its effect on cell proliferation.

<p>(<b>A</b>) Western blot showing the effect of EZH2 siRNA (si+) or a scrambled control siRNA (si-) on EZH2 protein levels in T265 and 90-8TL at 48h and 72 h post-transfection. (<b>B</b>) Cell proliferation monitored in time after transfection of T265 and 90-8TL with <i>EZH2</i> siRNA (si+) or a scrambled control siRNA (si-). ẞ-actin levels are shown as a loading control.</p

Expression level of <i>EZH2</i> in human neurofibroma and MPNST samples and cell lines.

<p>(<b>A</b>) qRT-PCR was used to determine mRNA levels of <i>EZH2</i> in paired plexiform neurofibroma (NF, blue, n = 9) and MPNST (red, n = 9) formalin-fixed paraffin-embedded tumor samples, each pair being derived from the same NF1 patient. Asterisk indicates P<0.05. (<b>B</b>) qRT-PCR was used to determine mRNA levels of <i>EZH2</i> in fresh frozen MPNST (red, n = 11), plexiform neurofibroma (blue, n = 7) and atypical neurofibroma (grey, n = 4). (<b>C</b>) qRT-PCR was used to determine mRNA levels of <i>EZH2</i> in a cell line panel: Hs53.T neurofibroma cell line (blue) and STS26T, sNF96.2, ST88-14, T265 and 90-8TL MPNST cell lines (red). (<b>D</b>) Western blot displaying EZH2 protein expression in cell line panel and HEK293T. ẞ-actin levels are shown as a loading control.</p

Sensitivity of neurofibroma and MPNST cell lines to doxorubucin.

<p>(<b>A</b>) An <i>in vitro</i> cytotoxicity assay (SRB assay) was used to determine IC₅₀ values (ng/ml) for doxorubucin of neurofibroma and MPNST cell lines after a 48h exposure to the drug. Graphs show cell viability as a function of doxorubucin concentration. Depicted is the average viability (n = 4) of a representative experiment. (<b>B</b>) Listing of calculated IC₅₀ values and correlation plot, with TOP2A protein expression levels on the Y-axis and IC₅₀ values for doxorubicin on the X-axis. Pearson correlation coefficient is depicted in the graph.</p

Sensitivity of neurofibroma and MPNST cell lines to the BET bromodomain inhibitor JQ1.

<p>(<b>A</b>) An <i>in vitro</i> cytotoxicity assay (SRB assay) was used to determine IC₅₀ values (nM) for the BET bromodomain inhibitor JQ1 of neurofibroma and MPNST cell lines after a 72h exposure to the drug. Graphs show cell viability as a function of JQ1 concentration. Depicted is the average viability (n = 4) of a representative experiment. (<b>B</b>) Listing of calculated IC₅₀ values and correlation plot, with BRD4 protein expression levels on the Y-axis and IC₅₀ values for JQ1 on the X-axis. Pearson correlation coefficient is depicted in the graph.</p

Expression level of <i>BRD4</i> in human neurofibroma and MPNST samples and cell lines.

<p>(<b>A</b>) qRT-PCR was used to determine mRNA levels of <i>BRD4</i> in paired plexiform neurofibroma (NF, blue, n = 9) and MPNST (red, n = 9) formalin-fixed paraffin-embedded tumor samples, each pair being derived from the same NF1 patient. Asterisk indicates P<0.05. (<b>B</b>) qRT-PCR was used to determine mRNA levels of <i>BRD4</i> in fresh frozen MPNST (red, n = 11), plexiform neurofibroma (blue, n = 7) and atypical neurofibroma (grey, n = 4). (<b>C</b>) qRT-PCR was used to determine mRNA levels of <i>BRD4</i> in a cell line panel: Hs53.T neurofibroma cell line (blue) and STS26T, sNF96.2, ST88-14, T265 and 90-8TL MPNST cell lines (red) (<b>D</b>) Western blot displaying BRD4 protein expression in cell line panel and HEK293T. ẞ-actin levels are shown as a loading control.</p