E2F3 is responsible for frequent amplification of 6p22.3 in human bladder cancer

The 6p22 is generally regarded as one of the most important amplification sites in urinary bladder cancer. Investigations, encouraged by these findings, subsequently lead to the delineation of the amplicon. During this process the genomic region was narrowed down to 1.7 Mb at 6p22.3, including presumably 13 different genes. Some of these genes were withdrawn from additional investigations due to low-level or absent expression in 6p22.3- amplified bladder cancers. Two genes, however, showed unquestionable correlation between high-level amplification and subsequent overexpression. But the relevant target gene that drives the amplification remained unidentified, yet. This work was ultimately aimed in a comprehensive comparison of the two remaining candidate oncogenes. The major findings were: - By performing FISH on a large bladder cancer TMA we show that NM_017774 is amplified in 11.6% of 893 tested human bladder cancer samples. Thus, the gene reaches an amplification level that is comparable to E2F3. - Following case-by-case re-evaluation of a large-section FISH analysis, exhibiting 104 6p22.3-amplified bladder cancers, demonstrates that both genes are 100% coamplified. - Furthermore, we show that both candidate oncogenes are always co-overexpressed in 6p22.3-amplified bladder cancer cell lines, presumably as a consequence of the amplification. - Experimentally decreased expression levels of NM_017774 and/or E2F3 similarly lead to strongly inhibited cell proliferation (observed in normal bladder cancer cells CRL-7930; without 6p22.3-amplification). - This finding suggests that NM_017774 -the gene of hitherto unknown function –must be functionally connected to the cell cycle regulatory machinery. - Besides, decreased E2F3-expression results in proliferation-reduction, and thus confirms the previously predicted essential role of this transcriptionfactor in cell cycle progression. - Finally, functional analysis performed in the 6p22.3-amplified HTB-5 cell line, demonstrate that E2F3 –but not NM_017774 -captures a limiting role for enhanced cellular proliferation in 6p22.3-amplified bladder cancer cells. - Hence, our results suggest that NM_017774 is only accidentally co-amplified because of its spatial neighbourhood to E2F3 (like other genes in the area), but does not have a functional role in 6p22.3-amplification, whatsoever. Conclusively, the findings of this study consistently document that amplification of 6p22.3 leads to upregulated mRNA expression, and increased protein production of the transcriptionfactor E2F3. While also other genes localized in the amplified region may be coamplified and co-overexpressed as a by-product of the amplification, E2F3 represents the main target gene and is therefore responsible for the frequent amplification of 6p22.3 in urinary bladder cancer

Ceramic foam plates: a new tool for processing fresh radical prostatectomy specimens

Procurement of fresh tissue of prostate cancer is critical for biobanking and generation of xenograft models as an important preclinical step towards new therapeutic strategies in advanced prostate cancer. However, handling of fresh radical prostatectomy specimens has been notoriously challenging given the distinctive physical properties of prostate tissue and the difficulty to identify cancer foci on gross examination. Here, we have developed a novel approach using ceramic foam plates for processing freshly cut whole mount sections from radical prostatectomy specimens without compromising further diagnostic assessment. Forty-nine radical prostatectomy specimens were processed and sectioned from the apex to the base in whole mount slices. Putative carcinoma foci were morphologically verified by frozen section analysis. The fresh whole mount slices were then laid between two ceramic foam plates and fixed overnight. To test tissue preservation after this procedure, formalin-fixed and paraffin-embedded whole mount sections were stained with hematoxylin and eosin (H&E) and analyzed by immunohistochemistry, fluorescence, and silver in situ hybridization (FISH and SISH, respectively). There were no morphological artifacts on H&E stained whole mount sections from slices that had been fixed between two plates of ceramic foam, and the histological architecture was fully retained. The quality of immunohistochemistry, FISH, and SISH was excellent. Fixing whole mount tissue slices between ceramic foam plates after frozen section examination is an excellent method for processing fresh radical prostatectomy specimens, allowing for a precise identification and collection of fresh tumor tissue without compromising further diagnostic analysis

¿CUÁNTO PESA UNA CÉLULA?

Tan sólo una célula ¿Cuánto pesa una célula humana? En octubre 2018,investigadores en Suiza dieron a conocer esta pequeña balanza en voladizodiseñada para detectar las más mínimas fluctuaciones en la masa de una célula viva

Role of KCNMA1 in breast cancer

KCNMA1 encodes the α-subunit of the large conductance, voltage and Ca(2+)-activated (BK) potassium channel and has been reported as a target gene of genomic amplification at 10q22 in prostate cancer. To investigate the prevalence of the amplification in other human cancers, the copy number of KCNMA1 was analyzed by fluorescence-in-situ-hybridization (FISH) in 2,445 tumors across 118 different tumor types. Amplification of KCNMA1 was restricted to a small but distinct fraction of breast, ovarian and endometrial cancer with the highest prevalence in invasive ductal breast cancers and serous carcinoma of ovary and endometrium (3-7%). We performed an extensive analysis on breast cancer tissue microarrays (TMA) of 1,200 tumors linked to prognosis. KCNMA1 amplification was significantly associated with high tumor stage, high grade, high tumor cell proliferation, and poor prognosis. Immunofluorescence revealed moderate or strong KCNMA1 protein expression in 8 out of 9 human breast cancers and in the breast cancer cell line MFM223. KCNMA1-function in breast cancer cell lines was confirmed by whole-cell patch clamp recordings and proliferation assays, using siRNA-knockdown, BK channel activators such as 17ß-estradiol and the BK-channel blocker paxilline. Our findings revealed that enhanced expression of KCNMA1 correlates with and contributes to high proliferation rate and malignancy of breast cancer

Ceramic foam plates: a new tool for processing fresh radical prostatectomy specimens

Procurement of fresh tissue of prostate cancer is critical for biobanking and generation of xenograft models as an important preclinical step towards new therapeutic strategies in advanced prostate cancer. However, handling of fresh radical prostatectomy specimens has been notoriously challenging given the distinctive physical properties of prostate tissue and the difficulty to identify cancer foci on gross examination. Here, we have developed a novel approach using ceramic foam plates for processing freshly cut whole mount sections from radical prostatectomy specimens without compromising further diagnostic assessment. Forty-nine radical prostatectomy specimens were processed and sectioned from the apex to the base in whole mount slices. Putative carcinoma foci were morphologically verified by frozen section analysis. The fresh whole mount slices were then laid between two ceramic foam plates and fixed overnight. To test tissue preservation after this procedure, formalin-fixed and paraffin-embedded whole mount sections were stained with hematoxylin and eosin (H&E) and analyzed by immunohistochemistry, fluorescence, and silver in situ hybridization (FISH and SISH, respectively). There were no morphological artifacts on H&E stained whole mount sections from slices that had been fixed between two plates of ceramic foam, and the histological architecture was fully retained. The quality of immunohistochemistry, FISH, and SISH was excellent. Fixing whole mount tissue slices between ceramic foam plates after frozen section examination is an excellent method for processing fresh radical prostatectomy specimens, allowing for a precise identification and collection of fresh tumor tissue without compromising further diagnostic analysis

Proliferation of breast cancer cells.

<p>Proliferation rates of MCF7, T47D, MFM223 and PC3 in response to siRNA targeting KCNMA1, BK-channel blocker paxilline and 17<i>β</i>-estradiol (E2). Proliferation rates of MCF7 and T47D were significantly reduced by siRNA and paxilline. MFM223 did not show a similar reduction after RNAi and strongly increased the proliferation rate upon treatment with paxilline; * p<0.05, ** p<0.01, n.s. = not significant.</p

Amplification of KCNMA1 in tumours.

<p>FISH analysis in a multi-tumour TMA with 2445 specimens from 118 different tumour types. The amplification was restricted to a small fraction of breast cancer and rare cases of ovarian and endometrial cancer.</p

Expression of KCNMA1 (micrographs).

<p>Immunofluorescence on a frozen tumour tissue sample and cytospins of different cell lines. Stainings represent BK-specific antibody (green) and DAPI (blue). Positive KCNMA1-staining in a KCNMA1-amplified frozen breast cancer specimen (A), positive staining in KCNMA1 amplified MFM223 at low (B) and high (C) magnification, negative staining in MCF7 (D) and T47D (E) at low magnification, and positive staining of the KCNMA1 amplified prostate cancer cell line PC3 (F; positive control) shown at high magnification. Bar = 10 µm.</p

Cell lines.

<p>Cell lines originating (a) with frequent and (b) without expression of sex hormone receptors.</p

Amplification of KCNMA1 in cancer cell lines.

<p>Expression profiles of 26 cancer cell lines, derived from different tissue types (quantitative real-time PCR analysis). KCNMA1 amplified cell lines MFM223 and PC3 showed the highest expression.</p