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Johannes Gerdes - One of the best experts on this subject based on the ideXlab platform.
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ki 67 as a molecular target for therapy in an in vitro three dimensional model for ovarian cancer
Cancer Research, 2010Co-Authors: Ramtin Rahmanzadeh, Bettina Baronluhr, Jonathan P Celli, Imran Rizvi, Johannes Gerdes, Tayyaba HasanAbstract:Targeting molecular markers and pathways implicated in cancer cell growth is a promising avenue for developing effective therapies. Although the Ki-67 Protein (pKi-67) is a key marker associated with aggressively proliferating cancer cells and poor prognosis, its full potential as a therapeutic target has never before been successfully shown. In this regard, its nuclear localization presents a major hurdle because of the need for intracellular and intranuclear delivery of targeting and therapeutic moieties. Using a liposomally encapsulated construct, we show for the first time the specific delivery of a Ki-67–directed antibody and subsequent lighttriggered death in the human ovarian cancer cell line OVCAR-5. Photoimmunoconjugate-encapsulating liposomes (PICEL) were constructed from anti–pKi-67 antibodies conjugated to fluorescein 5(6)-isothiocyanate, as a photoactivatable agent, followed by encapsulation in noncationic liposomes. Nucleolar localization of the PICELs was confirmed by confocal imaging. Photodynamic activation with PICELs specifically killed pKi-67– positive cancer cells both in monolayer and in three-dimensional (3D) cultures of OVCAR-5 cells, with the antibody TuBB-9 targeting a physiologically active form of pKi-67 but not with MIB-1, directed to a different epitope. This is the first demonstration of (a) the exploitation of Ki-67 as a molecular target for therapy and (b) specific delivery of an antibody to the nucleolus in monolayer cancer cells and in an in vitro 3D model system. In view of the ubiquity of pKi-67 in proliferating cells in cancer and the specificity of targeting in 3D multicellular acini, these findings are promising and the approach merits further investigation. Cancer Res; 70(22); 9234–42. ©2010 AACR.
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chromophore assisted light inactivation of pki 67 leads to inhibition of ribosomal rna synthesis
Cell Proliferation, 2007Co-Authors: Ramtin Rahmanzadeh, Johannes Gerdes, Gereon Huttmann, Thomas ScholzenAbstract:Objectives : Expression of the nuclear Ki-67 Protein (pKi-67) is strongly associated with cell proliferation. For this reason, antibodies against this Protein are widely used as prognostic tools for the assessment of cell proliferation in biopsies from cancer patients. Despite this broad application in histopathology, functional evidence for the physiological role of pKi-67 is still missing. Recently, we proposed a function of pKi-67 in the early steps of ribosomal RNA (rRNA) synthesis. Here, we have examined the involvement of pKi-67 in this process by photochemical inhibition using chromophore- assisted light inactivation (CALI). Materials and methods : Anti-pKi-67 antibodies were labelled with the fluorochrome fluorescein 5(6)-isothiocyanate and were irradiated after binding to their target Protein. Results : Performing CALI in vitro on cell lysates led to specific cross-linking of pKi-67. Moreover, the upstream binding factor (UBF) necessary for rRNA transcription was also partly subjected to cross-link formation, indicat- ing a close spatial proximity of UBF and pKi-67. CALI in living cells, using micro- injected antibody, caused a striking relocalization of UBF from foci within the nucleoli to spots located at the nucleolar rim or within the nucleoplasm. pKi-67-CALI resulted in dramatic inhibition of RNA polymerase I-dependent nucleolar rRNA synthesis, whereas RNA polymerase II-dependent nucleoplasmic RNA synthesis remained almost unaltered. Conclusions : Our data presented here argue for a crucial role of pKi-67 in RNA polymerase I-dependent nucleolar rRNA synthesis.
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ki 67 Protein is associated with ribosomal rna transcription in quiescent and proliferating cells
Journal of Cellular Physiology, 2006Co-Authors: Jo Ullwinkel, Johannes Gerdes, Ettina Aronluh, Anja Ludema, Claudia Wohlenberg, Thomas ScholzeAbstract:The nuclear Ki-67 Protein (pKi-67) has previously been shown to be exclusively expressed in proliferating cells. As a result, antibodies against this Protein are widely used as prognostic tools in cancer diagnostics. Here we show, that despite the strong downregulation of pKi-67 expression in non-proliferating cells, the Protein can nevertheless be detected at sites linked to ribosomal RNA (rRNA) synthesis. Although this finding does not argue against the use of pKi-67 as a proliferation marker, it has wide ranging implications for the elucidation of pKi-67 function. Employing the novel antibody TuBB-9, we could further demonstrate that also in proliferating cells, a fraction of pKi-67 is found at sites linked to the rRNA transcription machinery during interphase and mitosis. Moreover, chromatin immunoprecipitation (ChIP) assays provided evidence for a physical association of pKi-67 with chromatin of the promoter and transcribed region of the rRNA gene cluster. These data strongly suggest a role for pKi-67 in the early steps of rRNA synthesis. © 2005 Wiley-Liss, Inc.
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the ki 67 Protein interacts with members of the heterochromatin Protein 1 hp1 family a potential role in the regulation of higher order chromatin structure
The Journal of Pathology, 2002Co-Authors: Thomas Scholzen, Claudia Wohlenberg, Ian G Cowell, Elmar Endl, Sjaak Van Der Sar, Johannes GerdesAbstract:The expression of the nuclear Protein Ki-67 (pKi-67) is strictly correlated with cell proliferation. Because of this, anti-Ki-67 antibodies can be used as operational markers to estimate the growth fraction of human neoplasia in situ. For a variety of tumours, the assessment of pKi-67 expression has repeatedly been proven to be of prognostic value for survival and tumour recurrence, but no cellular function has yet been ascribed to the Ki-67 Protein. This study shows that a C-terminal domain of pKi-67 (Kon21) is able to bind to all three members of the mammalian heterochromatin Protein 1 (HP1) family in vitro and in vivo. This interaction can be manipulated in living cells, as evidenced by ectopic expression of GFP-tagged HP1 Proteins in HeLa cells, which results in a dramatic relocalization of endogenous pKi-67. Taken together, the data presented in this study suggest a role for pKi-67 in the control of higher-order chromatin structure. Copyright © 2001 John Wiley & Sons, Ltd.
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ki 67 expression during rat liver regeneration after partial hepatectomy
Hepatology, 1997Co-Authors: Christiane Gerlach, Dima Y Sakkab, Thomas Scholzen, Rebecca Dasler, Malcolm R Alison, Johannes GerdesAbstract:Antibodies to the cell-cycle-associated Ki-67 Protein have been widely used for more than a decade as markers of proliferative cells. The prototype antibody Ki-67 reacted only with snap-frozen human tissue, but a novel antibody, MIB-1, was able to detect the Ki-67 antigen in paraffin wax-embedded human tissue. The ability of MIB-5, a novel antibody reactive with the rat equivalent Ki-67 Protein, to immunohistochemically detect cycling parenchymal and littoral cells in the regenerating rat liver is reported. Rats underwent a standard two-thirds partial hepatectomy (PH), and groups of three animals were killed at intervals for up to 192 hours after PH. DNA synthesis was monitored by flash labeling with bromodeoxyuridine, and the response was as expected with a significant upsurge in hepatocyte labeling at 16 to 17 hours after PH. On the other hand, MIB-5 labeled a relatively constant percentage of hepatocytes (4%-8%) during the first 16 hours after PH, before a large proportion became labeled, also at 17 hours. The temporal pattern of MIB-5 labeling was similar to that of bromodeoxyuridine labeling, although, as expected, MIB-5 indices were higher. Semiquantification of Ki-67 messenger RNA (mRNA) levels by reverse-transcription polymerase chain reaction showed modest (fourfold to fivefold) increases in abundance during the first 12 hours after PH, but then levels increased dramatically to be at least 15-fold those of intact liver at 36 hours after PH. Much higher than normal levels of Ki-67 mRNA persisted throughout the period of study and even at 96 hours after PH they were still ninefold greater than normal. This study has shown the usefulness of the MIB-5 antibody to monitor proliferation in the rat liver, and furthermore, the pattern of expression of both the mRNA and the Protein suggest that the Ki-67 Protein, with hitherto unknown function, is more abundant in DNA synthesis and mitosis than in the early or even very late first G1 phase.
Mothaffar F Rimawi - One of the best experts on this subject based on the ideXlab platform.
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randomized phase ii study evaluating palbociclib in addition to letrozole as neoadjuvant therapy in estrogen receptor positive early breast cancer pallet trial
Journal of Clinical Oncology, 2019Co-Authors: Stephen S Johnston, Shannon Puhalla, Duncan Wheatley, Alistair Ring, Peter Barry, Chris Holcombe, Jean Francois Boileau, Louise Provencher, Andre Robidoux, Mothaffar F RimawiAbstract:PURPOSECDK4/6 inhibitors are used to treat estrogen receptor (ER)–positive metastatic breast cancer (BC) in combination with endocrine therapy. PALLET is a phase II randomized trial that evaluated the effects of combination palbociclib plus letrozole as neoadjuvant therapy.PATIENTS AND METHODSPostmenopausal women with ER-positive primary BC and tumors greater than or equal to 2.0 cm were randomly assigned 3:2:2:2 to letrozole (2.5 mg/d) for 14 weeks (A); letrozole for 2 weeks, then palbociclib plus letrozole to 14 weeks (B); palbociclib for 2 weeks, then palbociclib plus letrozole to 14 weeks (C); or palbociclib plus letrozole for 14 weeks. Palbociclib 125 mg/d was administered orally on a 21-days-on, 7-days-off schedule. Core-cut biopsies were taken at baseline and 2 and 14 weeks. Coprimary end points for letrozole versus palbociclib plus letrozole groups (A v B + C + D) were change in Ki-67 (Protein encoded by the MKI67 gene; immunohistochemistry) between baseline and 14 weeks and clinical response (ord...
Ramtin Rahmanzadeh - One of the best experts on this subject based on the ideXlab platform.
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a light controlled switch after dual targeting of proliferating tumor cells via the membrane receptor egfr and the nuclear Protein ki 67
Scientific Reports, 2016Co-Authors: Thomas Scholzen, Tayyaba Hasan, Gereon Huttmann, Sijia Wang, Zhenxi Zhang, Alfred Vogel, Ramtin RahmanzadehAbstract:Using nanotechnology for optical manipulation of molecular processes in cells with high spatial and temporal precision promises new therapeutic options. Especially tumor therapy may profit as it requires a combination of both selectivity and an effective cell killing mechanism. Here we show a dual targeting approach for selective and efficient light-controlled killing of cells which are positive for epidermal growth factor receptor (EGFR) and Ki-67. Liposomes with the covalently linked EGFR antibody Erbitux enabled selective uptake of FITC-labeled Ki-67 antibody TuBB-9 in EGFR-positive cells pre-loaded with the photoactive dye BPD. After irradiation at 690 nm, BPD disrupted the endosomal membranes and delivered the antibodies to the nucleoli of the cells. The second irradiation at 490 nm activated the FITC-labeled TuBB-9, which caused inactivation of the Ki-67 Protein and subsequent cell death via apoptosis. Efficient cell killing was possible at nanomolar concentrations of TuBB-9 due to the effective transport by immune liposomes and the high efficacy of the Ki-67 light-inactivation. Delivery of the liposomal constructs and cell destruction correlated well with the EGFR expression pattern of different cell lines (HeLa, OVCAR-5, MCF-7, and human fibroblasts), demonstrating an excellent selectivity.
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ki 67 as a molecular target for therapy in an in vitro three dimensional model for ovarian cancer
Cancer Research, 2010Co-Authors: Ramtin Rahmanzadeh, Bettina Baronluhr, Jonathan P Celli, Imran Rizvi, Johannes Gerdes, Tayyaba HasanAbstract:Targeting molecular markers and pathways implicated in cancer cell growth is a promising avenue for developing effective therapies. Although the Ki-67 Protein (pKi-67) is a key marker associated with aggressively proliferating cancer cells and poor prognosis, its full potential as a therapeutic target has never before been successfully shown. In this regard, its nuclear localization presents a major hurdle because of the need for intracellular and intranuclear delivery of targeting and therapeutic moieties. Using a liposomally encapsulated construct, we show for the first time the specific delivery of a Ki-67–directed antibody and subsequent lighttriggered death in the human ovarian cancer cell line OVCAR-5. Photoimmunoconjugate-encapsulating liposomes (PICEL) were constructed from anti–pKi-67 antibodies conjugated to fluorescein 5(6)-isothiocyanate, as a photoactivatable agent, followed by encapsulation in noncationic liposomes. Nucleolar localization of the PICELs was confirmed by confocal imaging. Photodynamic activation with PICELs specifically killed pKi-67– positive cancer cells both in monolayer and in three-dimensional (3D) cultures of OVCAR-5 cells, with the antibody TuBB-9 targeting a physiologically active form of pKi-67 but not with MIB-1, directed to a different epitope. This is the first demonstration of (a) the exploitation of Ki-67 as a molecular target for therapy and (b) specific delivery of an antibody to the nucleolus in monolayer cancer cells and in an in vitro 3D model system. In view of the ubiquity of pKi-67 in proliferating cells in cancer and the specificity of targeting in 3D multicellular acini, these findings are promising and the approach merits further investigation. Cancer Res; 70(22); 9234–42. ©2010 AACR.
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chromophore assisted light inactivation of pki 67 leads to inhibition of ribosomal rna synthesis
Cell Proliferation, 2007Co-Authors: Ramtin Rahmanzadeh, Johannes Gerdes, Gereon Huttmann, Thomas ScholzenAbstract:Objectives : Expression of the nuclear Ki-67 Protein (pKi-67) is strongly associated with cell proliferation. For this reason, antibodies against this Protein are widely used as prognostic tools for the assessment of cell proliferation in biopsies from cancer patients. Despite this broad application in histopathology, functional evidence for the physiological role of pKi-67 is still missing. Recently, we proposed a function of pKi-67 in the early steps of ribosomal RNA (rRNA) synthesis. Here, we have examined the involvement of pKi-67 in this process by photochemical inhibition using chromophore- assisted light inactivation (CALI). Materials and methods : Anti-pKi-67 antibodies were labelled with the fluorochrome fluorescein 5(6)-isothiocyanate and were irradiated after binding to their target Protein. Results : Performing CALI in vitro on cell lysates led to specific cross-linking of pKi-67. Moreover, the upstream binding factor (UBF) necessary for rRNA transcription was also partly subjected to cross-link formation, indicat- ing a close spatial proximity of UBF and pKi-67. CALI in living cells, using micro- injected antibody, caused a striking relocalization of UBF from foci within the nucleoli to spots located at the nucleolar rim or within the nucleoplasm. pKi-67-CALI resulted in dramatic inhibition of RNA polymerase I-dependent nucleolar rRNA synthesis, whereas RNA polymerase II-dependent nucleoplasmic RNA synthesis remained almost unaltered. Conclusions : Our data presented here argue for a crucial role of pKi-67 in RNA polymerase I-dependent nucleolar rRNA synthesis.
Fenghou Gao - One of the best experts on this subject based on the ideXlab platform.
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usp7 promotes cell proliferation through the stabilization of ki 67 Protein in non small cell lung cancer cells
The International Journal of Biochemistry & Cell Biology, 2016Co-Authors: Chao Zhang, Quanwu Zhang, Wei Zhao, Jiahui Guo, Shanling Liu, Bin Jiang, Fenghou GaoAbstract:The Ki-67 antigen (Ki-67) is the most reliable immunohistochemical marker for evaluation of cell proliferation in non-small cell lung cancer. However, the mechanisms underlying the regulation of Protein levels of Ki-67 in non-small cell lung cancer have remained elusive. In this study, we found that Ki-67 and ubiquitin-specific processing protease 7 (USP7) Protein were highly expressed in the nucleus of non-small cell lung cancer cells. Furthermore, statistical analysis uncovered the existence of a strong correlation between Ki-67 and USP7 levels. We could also show that the Protein levels of Ki-67 in non-small cell lung cancer cells significantly decreased after treatment with P22077, a selective chemical inhibitor of USP7, while the Ki-67 mRNA levels were unperturbed. Similar results were obtained by knocking down USP7 using short hairpin RNA (shRNA) in lung cancer cells. Interestingly, we noticed that ubiquitination levels of Ki-67 increased dramatically in USP7-silenced cells. The tests in vitro and vivo showed a significant delay in tumor cell growth upon knockdown of USP7. Additionally, drug sensitivity tests indicated that USP7-silenced A549 cells had enhanced sensitivity to paclitaxel and docetaxel, while there was no significant change in sensitivity toward carboplatin and cisplatin. Taken together, these data strongly suggest that the overexpression of USP7 might promote cell proliferation by deubiquitinating Ki-67 Protein, thereby maintaining its high levels in the non-small cell lung cancer. Our study also hints potential for the development of deubiquitinase-based therapies, especially those targeting USP7 to improve the condition of patients diagnosed with non-small cell lung cancer.
Stephen S Johnston - One of the best experts on this subject based on the ideXlab platform.
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randomized phase ii study evaluating palbociclib in addition to letrozole as neoadjuvant therapy in estrogen receptor positive early breast cancer pallet trial
Journal of Clinical Oncology, 2019Co-Authors: Stephen S Johnston, Shannon Puhalla, Duncan Wheatley, Alistair Ring, Peter Barry, Chris Holcombe, Jean Francois Boileau, Louise Provencher, Andre Robidoux, Mothaffar F RimawiAbstract:PURPOSECDK4/6 inhibitors are used to treat estrogen receptor (ER)–positive metastatic breast cancer (BC) in combination with endocrine therapy. PALLET is a phase II randomized trial that evaluated the effects of combination palbociclib plus letrozole as neoadjuvant therapy.PATIENTS AND METHODSPostmenopausal women with ER-positive primary BC and tumors greater than or equal to 2.0 cm were randomly assigned 3:2:2:2 to letrozole (2.5 mg/d) for 14 weeks (A); letrozole for 2 weeks, then palbociclib plus letrozole to 14 weeks (B); palbociclib for 2 weeks, then palbociclib plus letrozole to 14 weeks (C); or palbociclib plus letrozole for 14 weeks. Palbociclib 125 mg/d was administered orally on a 21-days-on, 7-days-off schedule. Core-cut biopsies were taken at baseline and 2 and 14 weeks. Coprimary end points for letrozole versus palbociclib plus letrozole groups (A v B + C + D) were change in Ki-67 (Protein encoded by the MKI67 gene; immunohistochemistry) between baseline and 14 weeks and clinical response (ord...
