The 3' Untranslated Region of the Cyclin B mRNA Is Not Sufficient to Enhance the Synthesis of Cyclin B during a Mitotic Block in Human Cells

<div><p>Antimitotic agents are frequently used to treat solid tumors and hematologic malignancies. However, one major limitation of antimitotic approaches is mitotic slippage, which is driven by slow degradation of cyclin B during a mitotic block. The extent to which cyclin B levels decline is proposed to be governed by an equilibrium between cyclin B synthesis and degradation. It was recently shown that the 3' untranslated region (UTR) of the murine cyclin B mRNA contributes to the synthesis of cyclin B during mitosis in murine cells. Using a novel live-cell imaging-based technique allowing us to study synthesis and degradation of cyclin B simultaneously at the single cell level, we tested here the role of the human cyclin B 3'UTR in regulating cyclin B synthesis during mitosis in human cells. We observed that the cyclin B 3'UTR was not sufficient to enhance cyclin B synthesis in human U2Os, HeLa or hTERT RPE-1 cells. A better understanding of how the equilibrium of cyclin B is regulated in mitosis may contribute to the development of improved therapeutic approaches to prevent mitotic slippage in cancer cells treated with antimitotic agents.</p> </div

Degradation and synthesis of the double-chimeric cyclin B reporter throughout the cell cycle by live-cell imaging at the single-cell level.

<p>(A) Degradation of the reporter is reflected by a decrease in BG430 fluorescence intensity at the metaphase to anaphase transition following pulse-chase labeling. (B) Whole protein expression of the CYS reporter molecule is reflected by YFP fluorescence intensity throughout the cell cycle. (C) An overlay of YFP (green), BG430 (blue) and mCherry (red) fluorescence is depicted by the lower image series. (D) Whole protein expression, degradation and synthesis/accumulation of the CYS reporter and pulse-chase labeling with the SNAP substrate is depicted for the indicated cell cycle stages by overlaying of YFP and BG430 fluorescence. (E) YFP (green curve) and BG430 (blue curve) fluorescence intensity curves are shown and the corresponding cell cycle stages are indicated (see cell cycle stage between the dotted lines). Background fluorescence was subtracted and the initial fluorescence intensity level was set to 100%. (F) A curve displaying the synthesis rate/accumulation of the cyclin B reporter (as defined by the difference between YFP- and BG430-fluorescence) is indicated.</p

Monitoring whole protein expression and degradation of the CYS reporter in the presence of spontaneous chromosomal misalignment in mitosis in U2Os cells.

<p>(A) Degradation of the reporter is reflected by a decrease in BG430 fluorescence intensity in a single cell showing misalignment of single chromosomes (white arrowheads, chromosomes appear in red). (B) Whole protein expression of the cyclin B reporter molecule is reflected by YFP fluorescence intensity. (C) Overlay of YFP and BG430 fluorescence. (D) YFP and BG430 fluorescence intensity curves for the cell depicted in A, B and C during mitosis (upper diagram). Fluorescence intensities during the metaphase delay are indicated as an enlargement in the lower diagram (enlarged section of the upper curve is indicated by the red dotted line).</p

Characterization of cyclin B kinetics in the presence of the cyclin B 3’ UTR in human tumor- and hTERT-immortalized cells and in the presence of the microtubule-stabilizer taxol.

<p>YFP and BG430 fluorescence intensity curves as determined by live-cell imaging of U2Os, HeLa and hTERT RPE-1 cells expressing the CYS reporter with a long 3’ UTR sequence. HeLa and hTERT RPE-1 cells that are arrested in a mitotic block using a final nocodazole concentration of 200ng/mL in the growth medium are depicted in (A) and (B) (mean fluorescence intensities +/- SD are indicated, n=4). U2Os, HeLa and hTERT RPE-1 cells that are arrested in a mitotic block using a final taxol concentration in the growth medium of 1µM are depicted in (C, D and E) (mean fluorescence intensities +/- SD are indicated, n=4). For every shown experimental setting the analyzed cells are representatives of three independent experiments each of which included at least 50.000 cells.</p

A double-chimeric system allowing the study of cyclin B kinetics at the single cell level in the presence and absence of the cyclin B 3'UTR.

<p>(A) A double-chimeric cyclin B reporter protein allowed the monitoring of whole protein expression by YFP fluorescence and degradation by pulse-chase labeling with the membrane-permeable SNAP substrate BG430. (B) Exemplary protein kinetics of the double-chimeric cyclin B reporter with GFP-SNAP were assessed by Western Blotting. Expression of the reporter protein and endogenous cyclin B following a nocodazole release are shown. (C) Plasmid map of the retroviral pLNCX2 vector indicating the sequence encoding the double-chimeric reporter and the 3' untranslated region (UTR). (D) Listing of different 3'UTRs found in available cyclin B full-length clones. (E) Sequence of the full-length 3'UTR indicating the sequence of the short 3'UTR variant in red and the hexanucleotide sequences (AATAAA) and the CPEs (T_4-5A_1-2T) in bold.</p

Intratibial Injection of Human Multiple Myeloma Cells in NOD/SCID IL-2Rγ(Null) Mice Mimics Human Myeloma and Serves as a Valuable Tool for the Development of Anticancer Strategies

<div><p>Background</p><p>We systematically analyzed multiple myeloma (MM) cell lines and patient bone marrow cells for their engraftment capacity in immunodeficient mice and validated the response of the resulting xenografts to antimyeloma agents.</p> <p>Design and Methods</p><p>Using flow cytometry and near infrared fluorescence in-vivo-imaging, growth kinetics of MM cell lines L363 and RPMI8226 and patient bone marrow cells were investigated with use of a murine subcutaneous bone implant, intratibial and intravenous approach in NOD/SCID, NOD/SCID treated with CD122 antibody and NOD/SCID IL-2Rγ(null) mice (NSG).</p> <p>Results</p><p>Myeloma growth was significantly increased in the absence of natural killer cell activity (NSG or αCD122-treated NOD/SCID). Comparison of NSG and αCD122-treated NOD/SCID revealed enhanced growth kinetics in the former, especially with respect to metastatic tumor sites which were exclusively observed therein. In NSG, MM cells were more tumorigenic when injected intratibially than intravenously. In NOD/SCID in contrast, the use of juvenile long bone implants was superior to intratibial or intravenous cancer cell injection. Using the intratibial NSG model, mice developed typical disease symptoms exclusively when implanted with human MM cell lines or patient-derived bone marrow cells, but not with healthy bone marrow cells nor in mock-injected animals. Bortezomib and dexamethasone delayed myeloma progression in L363- as well as patient-derived MM cell bearing NSG. Antitumor activity could be quantified via flow cytometry and in vivo imaging analyses.</p> <p>Conclusions</p><p>Our results suggest that the intratibial NSG MM model mimics the clinical situation of the disseminated disease and serves as a valuable tool in the development of novel anticancer strategies.</p> </div

A. Chemosensitivity of L363 cells in NSG against different antimyeloma agents determined by flow cytometry.

<p>A total of 24 NSG received it-injections of 2x10⁵ L363 cells: seven days thereafter, 18 mice were randomized into three groups of six animals each. Six additional mice were analysed on the first treatment day (d7) to determine tumor load before the respective therapies. MM cell engraftment was determined by flow cytometry using <i>h</i>CD138-Ab (n = 2-3 per group and time point, 4 measurements per group over the time). Black: mice were treated with control vehicle (0.9% NaCl, i.p. d7-11, 14-18). Yellow: mice were treated with 3 mg/kg/d dexamethasone, i.p. (d7-11, 14-18). Green: mice were treated with 0.7 mg/kg/d bortezomib, iv (d7, 11, 18). 7, 14, 28 and 35 days after implantation, tumor cells were assessed via flow cytometry and increased detectably. Analyses on day 35 confirmed that tumor growth was reduced with dexamethasone by 51% and with bortezomib by 66%. <b>B. Chemosensitivity of L363 cells in NSG against different antimyeloma agents determined by fluorescence-based-IVI.</b> A total of 15 NSG received it-injections of 2x10⁵ L363 cells: seven days thereafter, mice were randomized into two groups of six and nine animals, respectively. IVI using Alexa750 tagged <i>h</i>CD138 antibody was employed to determine tumor engraftment and treatment efficacy. In addition animals were x-rayed and the two pictures merged for optimal localization of the fluorescent region. Tumor growth after bortezomib treatment on day 18 was significantly reduced which was observed both for primary and metastatic sites (Student`s t-test, one-tailed p< 0.05). Error bars depict standard deviation. </p

A. Detection of human L363 and RPMI8226 cells in immunodeficient mice using <i>h</i>CD138 antibody labelled with Alexa750.

<p>20 mice per cell line were engrafted with L363 or RPMI8226 cells via different application routes. Images were taken once weekly for five weeks after implantation of the respective MM cell line with the Kodak Image Station <i>in </i><i>vivo</i>FX. In addition to the determination of tumor load via CCD camera, animals were x-rayed and the two pictures merged for optimal localization of the fluorescent region. No x-ray was performed for the animal bearing RPMI8226 in this figure. Intraosseal tumor growth was clearly detectable by the in vivo imaging (IVI) system (red circles). Additionally, BM metastases became apparent within the adjacent tibia and lumbar spine (yellow circles). <b>B. Detection of human L363 and RPMI8226 cells in immunodeficient mice using flow cytometry.</b> IVI data for L363 and RPMI8226 were confirmed by flow cytometry 35 days after tumor cell injection showing 42% and 22% human HLA-ABC and CD138 positive cells in L363 and 21% and 20% for RPMI8226, respectively. Analyses were performed on metastatic BM lesions. <b>C. Detection of human L363 and RPMI8226 cells in immunodeficient mice using immunohistochemistry.</b> IVI data for L363 and RPMI 8226 cells were confirmed by immunohistochemistry specific for human CD138+ cells (CD138+-infiltrates, red arrows) Analyses were performed on metastatic BM lesions.</p

Chemosensitivity of patient-derived MM cells in NSG against different antimyeloma agents determined by fluorescence-based-IVI (A-B) and flow cytometry (C) compared to clinical outcome of the donor patient (D).

<p>A total of 30 NSG received it-injections of 2x10⁶ patient derived MM cells: 11 days thereafter, mice were randomized into three groups of ten animals each. Black: mice were treated with control vehicle (0.9% NaCl, i.p. d11-15, 17-22). Yellow: mice were treated with 3 mg/kg/d dexamethasone, i.p. (d11-15, 17-22). Green: mice were treated with 0.7 mg/kg/d bortezomib, iv (d11, 15, 19, 22). <b>A. Tumor load determination using fluorescence based IVI.</b> IVI using Alexa750 tagged <i>h</i>CD138 antibody was employed to determine tumor engraftment and treatment efficacy from day 11 (first day of treatment) on once weekly. In addition animals were x-rayed and the two pictures merged for optimal localization of the fluorescent region. Non-tumor bearing animals were treated accordingly and used as negative control. Error bars depict standard deviation. Determination of net intensity over time revealed a reliable engraftment of patient-derived MM cells over time as compared to mock-injected mice. Treatment with bortezomib or dexamethasone reduced tumor progression compared to untreated control. Tumor growth after bortezomib and dexamethasone treatment on day 26 was significantly reduced by 40% and 22%, respectively, which was observed both for primary and metastatic sites (one way ANOVA, p<0.0001). <b>B. MM cell engraftment in the BM determined by flow cytometry.</b> MM cell engraftment in the BM was determined by flow cytometry using <i>h</i>CD138-Ab on the last experiment day (d=38, n=5 per group). Analyses confirmed that tumor growth was reduced with dexamethasone and bortezomib. <b>C. Immunhistochemical stainings of a femur from a NSG mouse inoculated with MM patient-derived BM cells.</b> Formalin fixed and paraffin embedded long bones from tumor bearing mice were stained with HE, anti-human CD38-, CD138- and kappa-Ab. The patient's kappa/CD38/CD138 triple-positive BM cells were well detectable in NSG mice 35 days after tumor cell inoculation. IHC analysis for hCD138 expression confirmed IVI and flow cytometry results as depicted in 6A+B. <b>D. Specific response parameters in the patient after treatment.</b> Specific response parameters in the patient after treatment. improved after three cycles of bortezomib, cyclophosphamide and dexamethasone (VCD) induction. A decline in his total protein (TP), gamma-globuline fraction (g-Gl), IgG, BM plasma cells (BM-PCs) and kappa-light chains (serum free light chains, [k-SFLC]) was induced as determinants of VCD response. Results are given as fold decreases from baseline levels.</p