Search

Ki-67 is one of the most famous marker proteins used by histologists to identify proliferating cells. Indeed, over 30 000 articles referring to Ki-67 are listed on PubMed. Here, we review some of the current literature regarding the protein. Despite its clinical importance, our knowledge of the molecular biology and biochemistry of Ki-67 is far from complete, and its exact molecular function(s) remain enigmatic. Furthermore, reports describing Ki-67 function are often contradictory, and it has only recently become clear that this proliferation marker is itself dispensable for cell proliferation. We discuss the unusual organization of the protein and its mRNA and how they relate to various models for its function. In particular, we focus on ways in which the intrinsically disordered structure of Ki-67 might aid in the assembly of the still-mysterious mitotic chromosome periphery compartment by controlling liquid-liquid phase separation of nucleolar proteins and RNAs.

The present study reports the synthesis of new purine bioisosteres comprising a pyrazolo[3,4-d]pyrimidine scaffold linked to mono-, di-, and trimethoxy benzylidene moieties through hydrazine linkages. First, in silico docking experiments of the synthesized compounds against Bax, Bcl-2, Caspase-3, Ki67, p21, and p53 were performed in a trial to rationalize the observed cytotoxic activity for the tested compounds. The anticancer activity of these compounds was evaluated in vitro against Caco-2, A549, HT1080, and Hela cell lines. Results revealed that two ( 5 and 7 ) of the three synthesized compounds ( 5 , 6 , and 7 ) showed high cytotoxic activity against all tested cell lines with IC 50 values in the micro molar concentration. Our in vitro results show that there is no significant apoptotic effect for the treatment with the experimental compounds on the viability of cells against A549 cells. Ki67 expression was found to decrease significantly following the treatment of cells with the most promising candidate: drug 7 . The overall results indicate that these pyrazolopyrimidine derivatives possess anticancer activity at varying doses. The suggested mechanism of action involves the inhibition of the proliferation of cancer cells.

Objective: Nucleus recognition is a critical yet challenging step in histopathology image analysis, for example, in Ki67 immunohistochemistry stained images. Although many automated methods have been proposed, most use a multi-stage processing pipeline to categorize nuclei, leading to cumbersome, low-throughput, and error-prone assessments. To address this issue, we propose a novel deep fully convolutional network for single-stage nucleus recognition., Methods: Instead of conducting direct pixel-wise classification, we formulate nucleus identification as a deep structured regression model. For each input image, it produces multiple proximity maps, each of which corresponds to one nucleus category and exhibits strong responses in central regions of the nuclei. In addition, by taking into consideration the nucleus distribution in histopathology images, we further introduce an auxiliary task, region of interest (ROI) extraction, to assist and boost the nucleus quantification with weak ROI annotation. The proposed network can be learned in an end-to-end, pixel-to-pixel manner for simultaneous nucleus detection and classification., Results: We have evaluated this network on a pancreatic neuroendocrine tumor Ki67 image dataset, and the experiments demonstrate that our method outperforms recent state-of-the-art approaches., Conclusion: We present a new, pixel-to-pixel deep neural network with two sibling branches for effective nucleus recognition and observe that learning with another relevant task, ROI extraction, can further boost individual nucleus localization and classification., Significance: Our method provides a clean, single-stage nucleus recognition pipeline for histopathology image analysis, especially a new perspective for Ki67 image quantification, which would potentially benefit individual object quantification in whole-slide images.

Background: The Ki-67 proliferation marker has multiple diagnostic and prognostic applications. Although several clones to the Ki-67 antigen are commercially available, the MIB1 clone is widely recommended in the surgical pathology literature for neuroendocrine tumors. In our cytopathology practice, we have encountered unexpectedly low MIB1 immunoreactivity in CytoLyt-fixed cell blocks (CBs). The current study evaluated the impact of fixatives, CB processing, and immunocytochemical (ICC) procedures on Ki-67 immunoreactivity., Methods: Test CBs were prepared from freshly resected tumors, and multiple variables in the MIB1 ICC procedure were tested, including CytoLyt versus formalin collection media, MIB1 versus other Ki-67 clones including 30-9, and other variables. MIB1 versus Ki-67 30-9 clones were tested in parallel on CytoLyt-fixed CBs from clinical samples of small cell lung carcinoma (SCLC)., Results: In the test CBs (n = 10), the mean MIB1 labeling index was 10% in CytoLyt versus 47% in formalin (P = .0116), with a mean loss of reactivity in matched CBs of 37% (up to 70%). None of the procedure modifications tested in 223 individual ICC reactions recovered MIB1 reactivity in CytoLyt except for switching to the Ki-67 30-9 antibody. In CytoLyt-fixed SCLC samples (n = 14), the Ki-67 30-9 antibody demonstrated expected ranges of reactivity (mean, 83%; range, 60%-100%), whereas MIB1 demonstrated markedly inhibited labeling (mean, 60%; range, 10%-95%) (P = .0058)., Conclusions: CytoLyt fixation substantially inhibits MIB1 immunoreactivity, whereas the Ki-67 30-9 clone is not susceptible to inhibition. Markedly discrepant MIB1 reactivity may present a pitfall in the diagnosis of SCLC and may lead to the incorrect prognostic stratification of other tumor types. For laboratories using CytoLyt, we recommend using the Ki-67 30-9 antibody rather than the MIB1 antibody., (© 2019 American Cancer Society.)

Pathologists have had increasing responsibility for quantitating immunohistochemistry (IHC) biomarkers with the expectation of high between-reader reproducibility due to clinical decision-making especially for patient therapy. Digital imaging-based quantitation of IHC clinical slides offers a potential aid for improvement; however, its clinical adoption is limited potentially due to a conventional field-of-view annotation approach. In this study, we implemented a novel solely morphology-based whole tumor section annotation strategy to maximize image analysis quantitation results between readers. We first compare the field-of-view image analysis annotation approach to digital and manual-based modalities across multiple clinical studies (~120 cases per study) and biomarkers (ER, PR, HER2, Ki-67, and p53 IHC) and then compare a subset of the same cases (~40 cases each from the ER, PR, HER2, and Ki-67 studies) using whole tumor section annotation approach to understand incremental value of all modalities. Between-reader results for each biomarker in relation to conventional scoring modalities showed similar concordance as manual read: ER field-of-view image analysis: 95.3% (95% CI 92.0-98.2%) vs digital read: 92.0% (87.8-95.8%) vs manual read: 94.9% (91.4-97.8%); PR field-of-view image analysis: 94.1% (90.3-97.2%) vs digital read: 94.0% (90.2-97.1%) vs manual read: 94.4% (90.9-97.2%); Ki-67 field-of-view image analysis: 86.8% (82.1-91.4%) vs digital read: 76.6% (70.9-82.2%) vs manual read: 85.6% (80.4-90.4%); p53 field-of-view image analysis: 81.7% (76.4-86.8%) vs digital read: 80.6% (75.0-86.0%) vs manual read: 78.8% (72.2-83.3%); and HER2 field-of-view image analysis: 93.8% (90.0-97.2%) vs digital read: 91.0 (86.6-94.9%) vs manual read: 87.2% (82.1-91.9%). Subset implementation and analysis on the same cases using whole tumor section image analysis approach showed significant improvement between pathologists over field-of-view image analysis and manual read (HER2 100% (97-100%), P=0.013 field-of-view image analysis and 0.013 manual read; Ki-67 100% (96.9-100%), P=0.040 and 0.012; ER 98.3% (94.1-99.5%), p=0.232 and 0.181; and PR 96.6% (91.5-98.7%), p=0.012 and 0.257). Overall, whole tumor section image analysis significantly improves between-pathologist's reproducibility and is the optimal approach for clinical-based image analysis algorithms.

Faithful segregation of the genetic material during the cell cycle is key for the continuation of life. Central to this process is the assembly of a bipolar spindle that aligns the chromosomes and segregates them to the two daughter cells. Spindle bipolarity is strongly dependent on the activity of the homotetrameric kinesin Eg5. However, another kinesin, Kif15, also provides forces needed to separate the spindle poles during prometaphase and to maintain spindle bipolarity at metaphase. Here we identify KBP as a specific interaction partner of Kif15 in mitosis. We show that KBP promotes the localization of Kif15 to the spindle equator close to the chromosomes. Both Kif15 and KBP are required for the alignment of all the chromosomes to the metaphase plate and the assembly of stable kinetochore fibers of the correct length. Taken together our data uncover a novel role for Kif15 in complex with KBP during mitosis.

Ki-67 and RepoMan have key roles during mitotic exit. Previously, we showed that Ki-67 organizes the mitotic chromosome periphery and recruits protein phosphatase 1 (PP1) to chromatin at anaphase onset, in a similar manner as RepoMan (Booth et al., 2014). Here we show how Ki-67 and RepoMan form mitotic exit phosphatases by recruiting PP1, how they distinguish between distinct PP1 isoforms and how the assembly of these two holoenzymes are dynamically regulated by Aurora B kinase during mitosis. Unexpectedly, our data also reveal that Ki-67 and RepoMan bind PP1 using an identical, yet novel mechanism, interacting with a PP1 pocket that is engaged only by these two PP1 regulators. These findings not only show how two distinct mitotic exit phosphatases are recruited to their substrates, but also provide immediate opportunities for the design of novel cancer therapeutics that selectively target the Ki-67:PP1 and RepoMan:PP1 holoenzymes., Competing Interests: The authors declare that no competing interests exist.

Introduction: Heat-induced epitope retrieval (HIER) of formalin-fixed paraffin-embedded tissues is now a standard practice in immunohistochemistry (IHC). In this study, we aimed to test the effect of altering HIER temperature on IHC staining quality at high altitude, the hypothesis being that lower HIER temperatures would result in improved staining patterns., Materials and Methods: In a laboratory at high altitude (Aurora, CO), we used a platform with automated onboard epitope retrieval, and systematically tested 3 different HIER temperatures (100°C, 95°C, 90°C) with 4 IHC stains that are commonly used in routine practice: CD3, Ki67, CK20, and Melan A (n=10 for each antibody/epitope retrieval temperature combination). A scoring system was devised, the slides were scored in a blinded manner, and statistical analysis was performed. For comparison, the same study was performed in a laboratory near sea level (Atlanta, GA)., Results: At high altitude, lower HIER temperatures resulted in improved staining patterns, as quantified by stronger staining intensity and greater area of the slides stained. The scores obtained with HIER temperatures of 95°C and 90°C were higher than those obtained with HIER of 100°C, and the difference was found to be statistically significantly for some antibody/epitope retrieval temperature combinations (P<0.05). This effect was not seen in the laboratory near sea level., Conclusions: We show that alternate epitope retrieval recommendations are warranted for laboratories at high altitude. Furthermore, we suggest that manufactures should consider how their instruments will perform at high altitude as they further automate the process of IHC.

Eukaryotic genomes are partitioned into chromosomes that form compact and spatially well-separated mechanical bodies during mitosis. This enables chromosomes to move independently of each other for segregation of precisely one copy of the genome to each of the nascent daughter cells. Despite insights into the spatial organization of mitotic chromosomes and the discovery of proteins at the chromosome surface, the molecular and biophysical bases of mitotic chromosome structural individuality have remained unclear. Here we report that the proliferation marker protein Ki-67 (encoded by the MKI67 gene), a component of the mitotic chromosome periphery, prevents chromosomes from collapsing into a single chromatin mass after nuclear envelope disassembly, thus enabling independent chromosome motility and efficient interactions with the mitotic spindle. The chromosome separation function of human Ki-67 is not confined within a specific protein domain, but correlates with size and net charge of truncation mutants that apparently lack secondary structure. This suggests that Ki-67 forms a steric and electrostatic charge barrier, similar to surface-active agents (surfactants) that disperse particles or phase-separated liquid droplets in solvents. Fluorescence correlation spectroscopy showed a high surface density of Ki-67 and dual-colour labelling of both protein termini revealed an extended molecular conformation, indicating brush-like arrangements that are characteristic of polymeric surfactants. Our study thus elucidates a biomechanical role of the mitotic chromosome periphery in mammalian cells and suggests that natural proteins can function as surfactants in intracellular compartmentalization.

The selective inhibition of intracellular and nuclear molecules such as Ki-67 holds great promise for the treatment of cancer and other diseases. However, the choice of the target protein and the intracellular delivery of the functional agent remain crucial challenges. Main hurdles are (a) an effective delivery into cells, (b) endosomal escape of the delivered agents, and (c) an effective, externally triggered destruction of cells. Here we show a light-controlled two-step approach for selective cellular delivery and cell elimination of proliferating cells. Three different cell-penetrating nano constructs, including liposomes, conjugates with the nuclear localization sequence (NLS), and conjugates with the cell penetrating peptide Pep-1, delivered the light activatable antibody conjugate TuBB-9-FITC, which targets the proliferation associated protein Ki-67. HeLa cells were treated with the photosensitizer benzoporphyrin monoacid derivative (BPD) and the antibody constructs. In the first optically controlled step, activation of BPD at 690 nm triggered a controlled endosomal escape of the TuBB-9-FITC constructs. In more than 75% of Ki-67 positive, irradiated cells TuBB-9-FITC antibodies relocated within 24 h from cytoplasmic organelles to the cell nucleus and bound to Ki-67. After a second light irradiation at 490 nm, which activated FITC, cell viability decreased to approximately 13%. Our study shows an effective targeting strategy, which uses light-controlled endosomal escape and the light inactivation of Ki-67 for cell elimination. The fact that liposomal or peptide-assisted delivery give similar results leads to the additional conclusion that an effective mechanism for endosomal escape leaves greater variability for the choice of the delivery agent.

Purpose: To investigate the relation between PET-CT SUVmax value and prognostic factors in locally advanced breast cancer., Methods: Data of 73 patients were retrospectively analyzed. Relations between SUVmax value, clinical stage, tumor grade and breast cancer molecular subtypes were analyzed by using one-way ANOVA and x(2) tests. Correlations between age, ki-67 scores and SUVmax were evaluated by using Pearson's correlation test. A p value <0.05 was considered statistically significant., Results: Median SUVmax values for clinical stages 1, 2 and 3 were 5 (range 2.1-4.1), 10.6 (range 2.9-19.6), and 12.2 (range 3.2-23.3), respectively. Statistically significant difference was noticed between stage 1 and 2 (p=0.014) and stage 1 and 3 (p=0.001). Median SUVmax values of triple negative, luminal A, luminal B and non-luminal HER2 positive groups were 14.4 (range 6.6-23.3), 8.2 (range 2.1-18.2), 10.1 (range 3.5-19.6), and 14 (range 4.1-22.9), respectively. Statistically significant differences were noticed in SUVmax values between triple-negative and luminal A groups (p=0.005) and between non-luminal HER2 positive and luminal A groups (p=0.02). Median SUVmax values of grade 1, 2 and 3 were 5.7 (range 2.1-18.2), 9.5 (range 2.2-21.3), and 11.6 (range 3.5-23), respectively. Statistically significant difference was noticed only between SUVmax values of grade 1 and 3 (p=0.035). There was negative correlation between age and SUVmax value (r=-0.23, p=0.047) and positive correlation between ki-67 and SUVmax value (r=0.43, p=0.016)., Conclusion: There were significant positive relations between PET-CT SUVmax value and clinical stage, tumor grade, and certain breast cancer molecular subtypes (triple-negative and non-luminal HER2 positive groups. Moreover, positive correlation was found between SUVmax value and ki-67 and negative correlation between SUVmax value and age.

Some report that adult human β-cells do not replicate, but we postulate this assumption is erroneous due a postmortem decline in replication markers such as Ki67. Our earlier report showed that Ki67-marked β-cells were rarely found in human cadaveric pancreases but were in the range of 0.2-0.5% in human islets transplanted into mice. This study subjected 4-week-old mice to autopsy conditions that typically occur with humans. Mice were killed, left at room temperature for 3 h, and then placed at 4°C for 3, 9, or 21 h. There was a rapid marked fall in Ki67 staining of β-cells compared with those fixed immediately. Values at death were 6.9 ± 0.9% (n = 6) after a 24-h fast, 4.1 ± 0.9% (n = 6) at 3 h room temperature, 2.7 ± 0.7% (n = 5) at 6 h, 1.6 ± 0.6% (n = 5) at 12 h, and 2.9 ± 0.8% (n = 5) at 24 h. Similar postmortem conditions in newborn pigs resulted in very similar declines in Ki67 staining of their β-cells. These data support the hypothesis that conclusions on the lack of replication of adult human β-cells are incorrect and suggest that adult human β-cells replicate at a low but quantitatively meaningful rate., (© 2015 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.)

Hotspot detection plays a crucial role in grading of neuroendocrine tumours of the digestive system. Hotspots are often detected manually from Ki-67-stained images, a practice which is tedious, irreproducible and error prone. We report a new method to segment Ki-67-positive nuclei from Ki-67-stained slides of neuroendocrine tumours. The method combines minimal graph cuts along with the multistate difference of Gaussians to detect the individual cells from images of Ki-67-stained slides. It, then, automatically defines the composite function, which is used to determine hotspots in neuroendocrine tumour slide images. We combine modified particle swarm optimization with message passing clustering to mimic the thought process of the pathologist during hotspot detection in neuroendocrine tumour slide images. The proposed method was tested on 55 images of size 10 × 5 K and resulted in an accuracy of 94.60%. The developed methodology can also be part of the workflow for other diseases such as breast cancer and glioblastomas., (© 2014 The Authors Journal of Microscopy © 2014 Royal Microscopical Society.)

Formalin-fixed tissue has been a mainstay of clinical pathology laboratories, but formalin alters many biomolecules, including nucleic acids and proteins. Meanwhile, frozen tissues contain better-preserved biomolecules, but tissue morphology is affected, limiting their diagnostic utility. Molecular fixatives promise to bridge this gap by simultaneously preserving morphology and biomolecules, enabling clinical diagnosis and molecular analyses on the same specimen. While previous reports have broadly evaluated the use of molecular fixative in various human tissues, we present here the first detailed assessment of the applicability of molecular fixative to both routine histopathological diagnosis and molecular analysis of cervical tissues. Ten specimens excised via the loop electrosurgical excision procedure, which removes conical tissue samples from the cervix, were cut into alternating pieces preserved in either formalin or molecular fixative. Cervical specimens preserved in molecular fixative were easily interpretable, despite featuring more eosinophilic cytoplasm and more recognizable chromatin texture than formalin-fixed specimens. Immunohistochemical staining patterns of p16 and Ki-67 were similar between fixatives, although Ki-67 staining was stronger in the molecular fixative specimens. The RNA of molecular fixative specimens from seven cases representing various dysplasia grades was assessed for utility in expression microarray analysis. Cluster analysis and scatter plots of duplicate samples suggest that data of sufficient quality can be obtained from as little as 50ng of RNA from molecular fixative samples. Taken together, our results show that molecular fixative may be a more versatile substitute for formalin, simultaneously preserving tissue morphology for clinical diagnosis and biomolecules for immunohistochemistry and gene expression analysis., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: 3,5,3'-Triiodothyronine (T3) has a stimulatory effect on cellular growth via thyroid hormone receptors (TRs) in several cell lines. TR expression in the pancreas suggests that pancreatic beta cell proliferation might be induced by T3. The purpose of this study was to demonstrate that T3 induces pancreatic beta cell proliferation through the mitogen activated protein kinase/extracellular regulated kinase (MAPK/ERK) pathway., Methods: INS-1 cells were plated as a monolayer at densities of 4×104, cultured in RPMI 1,640 with 10% fetal bovine serum with 2-mercaptoethanol, respectively, in 6-well multiplates. After 48 h, they were exposed to 10-7 M T3 or to vehicle alone. Viable cells were harvested after 24, 48, and 72 h of continuous exposure. Cell proliferation and TRα1 and TRβ1 expression were analyzed by flow-assisted cell sorting analysis, Ki-67 staining, and Western blotting. The p38 MAPK, ERK, and Akt pathways were analyzed by Western blotting. Beta cell function was evaluated by assaying insulin secretion., Results: T3 enhanced INS-1 cell proliferation at a dose of 10-7 M in a time-dependent manner via the MAPK/ERK pathway and promoted insulin secretion., Conclusions: Our results demonstrate that MAPK/ERK pathway plays an important role in the T3 induced pancreatic beta cell proliferation., (© J. A. Barth Verlag in Georg Thieme Verlag KG Stuttgart · New York.)

Objectives: Dysregulation of Met signalling is associated with malignant transformation. Combined treatment has been shown to reduce Met activation and mammary tumour cell proliferation. Experiments here, were conducted to determine mechanisms involved in mediating anti-cancer effects of combined γ-tocotrienol and SU11274 (Met inhibitor) treatment in various mammary cancer cell lines., Materials and Methods: Treatment effects on mouse (+SA) and human (MCF-7, and MDA-MB-231) mammary cancer cell lines, and normal mouse (CL-S1) and human (MCF10A) mammary epithelial cell lines were compared. Cell proliferation and survival were determined by MTT assay and Ki-67 staining; protein expression was determined by western blot analysis. Immunofluorescence staining was also used to characterize expression and localization of multiple epithelial and mesenchymal markers. Cell migration was determined using a wound-healing assay., Results: Combined treatment with γ-tocotrienol and SU11274 resulted in synergistic inhibition of +SA, MCF-7, and MDA-MB-231, but not CL-S1 or MCF10A cell growth that was associated with reduction in Akt STAT1/5 and NFκB activation and corresponding blockade in epithelial-to-mesenchymal transition, as indicated by increased expression of E-cadherin, β-catenin, and cytokeratins 8/18 (epithelial markers) and corresponding reduction in vimentin (mesenchymal marker) and reduction in cancer cell motility., Conclusions: Suggest that combined γ-tocotrienol and Met inhibitor treatment may provide benefit in treatment of breast cancers characterized by aberrant Met activity., (© 2013 John Wiley & Sons Ltd.)

The proliferative index in breast carcinoma is usually calculated by the percentage of the Ki-67 positive cells out of the total number of malignant cells. In order to reduce the inter-observer variability of the calculated proliferative index a cocktail of antibodies against E-Cadherin and Ki-67 (Ki/Cad Cocktail) is presented. The cocktail was applied on 59 cases of infiltrating duct carcinoma of breast and compared to the consecutive slides stained for Ki-67 alone. The Ki/Cad cocktail has the advantage that by adding the anti E-Cadherin antibody, all the malignant epithelial cells are highlighted and can be differentiated from other proliferating cells. Statistical analysis proved that the cocktail increases the inter-observer agreement from 89 % to 97 % as compared to the Ki-67 alone and also reduces the overlap between the cancer grades.

Objectives: To evaluate the CINtec PLUS assay (mtm laboratories), a new immunocytochemical method for the simultaneous detection of p16(INK4a) and Ki-67, in liquid-based cervico-vaginal cytology, investigating the association of the dual staining with HPV infection and genotyping as well as cytological and histological abnormalities., Methods: 140 women with a cervico-vaginal sample obtained immediately before the colposcopy were enrolled. This cytological sample was used for HPV testing with the Linear Array HPV Genotyping Test, the dual staining with the CINtec PLUS kit and the morphology assessment., Results: Cytology results were 38 NILM, 16 ASC-US, 32L-SIL, 54H-SIL or worse. 113 patients also had a colposcopy-guided biopsy, classified as 14 negative, 35 CIN1, 24 CIN2, 37 CIN3, 3 invasive SCC. A strong association between p16/Ki-67 and HR-HPV infection was found (COR=6.86, 95% CI: 1.84-31.14). Importantly, the association between p16/Ki-67 positivity and HPV16 and/or 18 infection was 2-fold stronger compared to that with the infection by other HR-HPV types (COR=9.92, 95% CI: 2.39-47.77 vs COR=4.20, 95% CI: 0.99-20.87). In addition, p16/Ki-67 positivity rate significantly increased with the severity of the cytological and histological abnormalities (p<0.05 in both cases). p16/Ki-67 positivity resulted strongly associated with a CIN2+ diagnosis (COR=10.86 95% CI: 4.16-29.12)., Conclusions: This preliminary study evidenced that p16/Ki-67 immunostaining might have a relevant clinical role, since the dual staining was significantly associated with HR-HPV infection, particularly with HPV 16 and 18, and the increasing grade of the cervical lesions, the positivity for this biomarker being strongly related to the presence of a CIN2+ lesion., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: Knowledge of the clinical efficacy in recurrent neuroendocrine carcinomas is sparse. Treatment with temozolomide alone or in combination with capecitabine and bevacizumab has recently shown promising results., Patients and Methods: Analysis of consecutive patients with neuroendocrine carcinomas (Ki-67 proliferation index >20%) and performance status 0-2 treated with temozolomide 200 mg/sqm orally days 1-5 every 28 days after at least one previous platin-containing chemotherapy regimen., Results: Twenty-eight eligible patients received a median of 3 courses. Sixteen patients were evaluable for response: Six achieved stable disease and ten progressed. The median survival for the 28 patients was 3.5 months. Survival in patients with tumors of pancreatic origin (n = 7) was 7.0 months versus 2.9 months in non-pancreatic origin (n = 21). Patients in PS 0-1 (n = 22) had a median survival of 4.5 months versus 1.1 months in patients in PS 2 (n = 6). Ki-67 index ≥ 50% was associated with a significantly shorter median survival than Ki-67 index <50% (2.7 months versus 10.9 months). The treatment was well tolerated., Conclusion: Temozolomide monotherapy has limited effect in treatment of recurrent neuroendocrine carcinomas. Second line treatment with temozolomide in combination with other compounds should be further investigated in patients in good performance with Ki-67 index <50%.

Objective: To investigate the relationships between suburothelial inflammation and urothelial dysfunction in interstitial cystitis/painful bladder syndrome (IC/PBlS)., Materials and Methods: Immunofluorescence staining of ki-67 (to assess cell proliferation), junction protein E-cadherin, tryptase (to assess mast cell activation) and TUNEL (to assess urothelial apoptosis) were performed in bladder tissues from 20 patients with IC/PBlS and from 6 control patients. The fluorescence intensity of E-cadherin was measured using the ImageJ method. The percentage of apoptotic cells, proliferated cells and activated mast cells were measured and quantified as positive cells (±SD) per area unit (4 µm(2))., Results: The ratio of ki-67-positive cells in the bladder tissue of the patients with IC/PBlS was significantly down-regulated compared with that of the control patients (0.559 ± 0.658 vs. 1.23 ± 1.28, P = 0.001). TUNEL staining revealed a significantly higher number of apoptotic cells in the IC/PBlS bladder tissue compared with control bladder tissue (2.26 ± 2.04 v 0.051 ± 0.124, P = 0.000). The tryptase signal was significantly stronger in the IC/PBlS bladder tissue compared with that of control patients (6.16 ± 4.35 v 1.15 ± 0.436, P = 0.000). The apoptotic cell number in IC/PBlS bladder tissue correlated significantly with mast cell activation (P = 0.021). Immunofluorescence also showed a significantly lower distribution of E-cadherin in IC/PBlS bladder tissue compared with that of control patients (8.50 ± 6.83 v 17.2 ± 11.9, P = 0.000). Lower expression of E-cadherin in IC/PBlS bladder tissue was significantly correlated with higher visual analogue pain scores in patients with IC/PBlS (P= 0.008)., Conclusions: The results of the present study suggest that urothelial homeostasis in IC/PBlS bladders was impaired, and abnormal urothelial function was significantly associated with chronic inflammation. The junctions between urothelial cells in IC/PBlS bladders were abnormal, which was associated with the patient's self-report pain scales., (© 2010 THE AUTHORS. BJU INTERNATIONAL © 2010 BJU INTERNATIONAL.)

Using explicit solvent molecular dynamics simulations, we were able to obtain direct observations of shifts in the hydrogen-bonding register of an intermolecular β-sheet protein-peptide complex. The β-sheet is formed between the FHA domain of cancer marker protein Ki67 (Ki67FHA) and a peptide fragment of the hNIFK signaling protein. Potential encounter complexes of the Ki67FHA receptor and hNIFK peptide are misregistered states of the β-sheet. Rearrangements of one of these misregistered states to the native state were captured in three independent simulations. All three rearrangements occurred by a common mechanism: an aromatic residue of the peptide (F263) anchors into a transient hydrophobic pocket of the receptor to facilitate the formation of native hydrogen bonds. To our knowledge, these simulations provide the first atomically detailed visualizations of a mechanism by which nature might correct for errors in the alignment of intermolecular β-sheets., (Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Objective: To present a computerized system for cell counting in histopathologic slides of meningioma and oligodendroglioma stained immunohistochemically against Ki-67 antigen and discuss the variability of tumor cell numbers in the field of view of analyzed neoplasms to standardize tumor cellularity., Study Design: A computer program using an algorithm based on mathematical morphology was developed to perform quantitative evaluation of slides. That solution was combined with the Support Vector Machine used for classification of cell immunoreactivity., Results: The mean number of cells in the analyzed field of view from patients with meningioma was 623. Of these, 95% were in the 386-781 cells range. In oligodendrogliomas the mean was 474 cells and all results were in the 204-736 range. The mean relative discrepancy between results of our system and human expert score was 8%., Conclusion: The proposed system appeared to be an efficient tool for supporting histopathologic diagnosis. The applied sequential thresholding simulated well the human process of cell recognition. Cellularity of the analyzed tumors did not show stability within the specimens from different patients. The results were also highly variable in different fields of view obtained from the same patient.

We investigated refolding processes of beta-sheeted protein FHA domains (FHA1 domain of Rad53 and Ki67 FHA domain) by cryo-stopped-flow (SF) method combined with far-ultraviolet (far-UV) circular dichroism (CD, the average secondary structure content) and small angle X-ray scattering (SAXS, measuring the radius of gyration). In case of FHA1 domain of Rad53, no detectable time course was observed except the initial burst on its refolding process at 4 degrees C, suggesting that the FHA1 domain of Rad53 was already refolded to its native state within the dead time of the SF apparatus and the rate of the refolding is too fast to be observed at this temperature. In contrast, there was an observable alpha-helical burst at -15 degrees C and -20 degrees C in the presence of 45% ethylene glycol (EGOH) by CD-SF. Besides, the radius of gyration (Rg) of the burst phase intermediate at -20 degrees C shows the intermediate is already compact, and the compaction process was accompanied with the decrease of alpha-helical content at the same temperature. In case of Ki67 FHA domain, ellipticity change at 222 nm was observed on its refolding pathway at -28 degrees C in the presence of 45% EGOH and 2 mM DTT, indicating that Ki67 FHA domain also takes non-native alpha-helix-rich intermediate on its folding pathway. Time-resolved SAXS experiment was done. As the signal/noise ratio is low, we could not observe the time-dependent signal change through the time course. However, the initial Rg value was obtained as 18.2 +/- 0.5 A, which is much smaller than the unfolded Rg value (26.5 +/- 1.2 A), and is slightly larger than the native one (15.9 +/- 1.8 A). These results suggest that Ki67 FHA domain also forms compact non-native alpha-helix-rich intermediate before refolding to its native beta-structure on the refolding pathway. These results are in good agreement with other beta-proteins, such as bovine beta-lactoglobulin (BLG), src SH3 domain proteins. It seems the alpha-helical burst phases appear on the folding pathway of beta-sandwiched proteins., (Copyright 2010 Elsevier Masson SAS. All rights reserved.)

Genetic based reporters have distinct advantages over classical immunocytochemical techniques for probing cellular functions. Most importantly, they enable dynamic real-time visualization and quantification of cellular processes in living cells and tissue. This study was conducted to generate a genetic based reporter to label cells that transitioned from the G(0) to G(1)/S phases of the cell cycle, hypothesizing that the proximal promoter of the Ki67 (Ki67p) gene, a commonly used cytology marker induced during this transition, would contain the suitable regulatory elements to drive marker gene expression. This study reports the cloning and characterization of the 1.5 kb proximal promoter (Ki67p) of the human Ki67 gene. Ki67p driven GFP expression colocalizes in cells with endogenous Ki67 expression and is correlated with cells transitioning through S/G(2)/M phases of the cell cycle. Treatment Ki67p-GFP expressing HT1080 cells with mitomycin C, an antineoplastic agent, induces P21 and P27 expression, G(1)/S/G(2)M block and attenuates Ki67p activity. Attenuation of the Ki67p also occurs during cell-density induced cell cycle arrest. Taken together, these results indicate that the Ki67p can be used to identify proliferating subpopulations of live cells in intact complex three-dimensional cellular aggregates, such as embryoid bodies, thus providing some unique advantages over conventional immunohistochemical approaches., (Copyright 2010 International Society for Advancement of Cytometry.)

In order to improve cell therapy techniques, we have characterized a multipotent neural precursor cell isolation technique from the subventricular zone of adult pig brain. The pig is a non-primate species that is immunologically closest to human. The proliferative zone of this neurogenic structure was first localized in situ in the pig brain by Ki-67 immunohistochemistry, as a ventral subfield of the Nissl-stained subventricular zone. For in vitro cultures, the striatal forebrain was sampled from deeply anaesthetized adult pigs and SVZ tissue explants were immediately microdissected out and dissociated in the appropriate medium. Primary cell culture in the presence of EGF and bFGF allowed growth of spherical masses that exhibited sustained growth and self-renewal capacity through six subsequent passages. Molecular characterization using reverse transcription-polymerase chain reaction (RT-PCR) showed that expanded pro-differentiating neurospheres expressed markers of proliferation, neural stem cells, and committed neural progenitors. After growth factor removal, the spheres became adherent and were shown to contain the three neural cell lineages by triple immunocytofluorescence and confocal microscopy. The present protocol therefore allowed for in vitro expansion of pig brain primary cells that display capacities for proliferation, self-renewal, and multipotency, i.e., the cardinal features of multipotent neural precursor cells.

Purpose: The aim was to investigate the potential impact of positron emission tomography (PET)/computed tomography (CT) as compared to PET and CT on the staging of patients with indolent lymphoma., Procedures: PET/CTs from 45 patients with indolent lymphoma undergoing staging or restaging were studied. Clinical follow-up, additional imaging, and histology served as the gold standard., Results: PET/CT correctly diagnosed 92 nodal regions as positive for lymphomatous involvement and 458 as disease free vs 68 and 449 for PET and 64 and 459 for CT, respectively. The respective sensitivities, specificities, and accuracies were 99%, 100%, and 99.8% for PET/CT, 68%, 97.5%, and 92.2% for PET, and 70%, 100%, and 94.7% for CT. PET/CT performed significantly better than PET (p < 0.001 for sensitivity, specificity, and accuracy) and CT (p < 0.001 for sensitivity and accuracy). PET/CT also correctly identified significantly more extra-nodal lesions (22) than CT (14) and PET (nine)., Conclusions: PET/CT provides significantly more accurate information compared to PET and CT for the staging and re-staging of patients with indolent lymphoma.

The forkhead-associated (FHA) domain of human Ki67 interacts with the human nucleolar protein hNIFK, recognizing a 44-residue fragment, hNIFK226-269, phosphorylated at Thr234. Here we show that high-affinity binding requires sequential phosphorylation by two kinases, CDK1 and GSK3, yielding pThr238, pThr234 and pSer230. We have determined the solution structure of Ki67FHA in complex with the triply phosphorylated peptide hNIFK226-269(3P), revealing not only local recognition of pThr234 but also the extension of the beta-sheet of the FHA domain by the addition of a beta-strand of hNIFK. The structure of an FHA domain in complex with a biologically relevant binding partner provides insights into ligand specificity and potentially links the cancer marker protein Ki67 to a signaling pathway associated with cell fate specification.

A cell proliferation marker protein, pKi-67, distributes to the chromosome periphery during mitosis and nucleolar heterochromatin in the interphase. We report here on the structural domains of pKi-67 that are required for its correct distribution. While both the LR domain and the conserved domain were involved in localization to the nucleolar heterochromatin, both the LR domain and the Ki-67 repeat domain were required for its distribution to the mitotic chromosome periphery. Using in vivo time-lapse microscopy, GFP-pKi-67 was dynamically tracked from the mitotic chromosome periphery to reforming nucleoli via prenucleolar bodies (PNBs). The signals in PNBs then moved towards and fused into the reforming nucleoli with a thin string-like fluorescence during early G1 phase. An analysis of the in vivo kinetics of pKi-67 using photobleaching indicated that the association of pKi-67 with chromatin was progressively altered from "loose" to "tight" after the onset of anaphase. These findings indicate that pKi-67 dynamically alters the nature of the interaction with chromatin structure during the cell cycle, which is closely related to the reformation process of the interphase nucleolar chromatin.

This study represents a combined lectin and immuno-histochemical analysis of normal, dysplastic and malignant squamous epithelia of the upper aerodigestive tract with emphasis on the relation between lectin reactivity and the cells' proliferative/anti-apoptotic potential. Staining by double-labeling to detect pKi67, DeltaNp63alpha, alpha2,3/6-linked sialic acid (NeuNAc) and galectin-3-reactive epitopes was performed on specimens of 8 normal cases, 27 primary tumours and 15 regional lymph node metastases. Normal epithelia expressed alpha2,6-linked NeuNAc in the basal layer, while alpha2,3-linked NeuNAc was observed in suprabasal layers. In normal epithelium pKi67 and DeltaNp63alpha positivity was seen in the basal layer and galectin-3-reactive sites in suprabasal layers. The studied squamous cell carcinomas and their metastases showed a tendency for stratification but with markedly altered architecture. The expression of the studied markers was heterogeneous between the different cancer cases but comparable between the corresponding primary and secondary lesions. Glycophenotypic properties were correlated with the level of differentiation. Tumour cell populations were characterized by occurrence of the p63+/pKi67+ alpha2,3-NeuNAc+ alpha2,6-NeuNAc+ phenotype. Analysis of the expression patterns of pKi67, p63 and galectin-3-reactive epitopes (Gal-3-RE) delineated statistically significant interrelationships (Gal-3-RE vs. p63: r = -0.709; Gal-3-RE vs. pKi67: r = -0.623; pKi67 vs. p63: r = 0.895). Of the studied markers only Gal-3-RE expression was correlated to the differentiation-dependent grading (G1 vs. G2: p = 0.007, G2 vs. G3: p = 0.006, G1 vs. G3: p = 0.002). DeltaNp63alpha expression was statistically different only between G1 and G3 (p = 0.03). No statistically significant differences were detected between the primary tumours and the corresponding regional lymph node metastases. Based on the concept of the sugar code further analysis of cell characteristics such as proliferation together with lectin histochemical features, especially using tissue lectins as probes, is thus warranted.

Objectives: The aim of this study was to evaluate the significance of p53 expression and proliferative activity of glandular epithelium and intestinal metaplasia in Helicobacter pylori associated gastritis of pediatric patients., Methods: The study included endoscopic gastric biopsies of 54 children with dyspeptic complaints. Immunohistochemistry was performed for evaluation of p53 expression and Ki-67 labeling index, an indicator of proliferative activity. Grading of H. pylori density, intestinal metaplasia and inflammatory cell infiltration were performed in histologic tissue sections stained with hematoxylin-eosin, Giemsa and Alcian-blue., Results: Of 54 children, 35 (64%) were infected by H. pylori. Positive immunostaining for p53 was observed in 11 of 54 cases (20.4%). H. pylori infection was found in 10 (91%) of the p53-positive patients. There was a positive correlation between H. pylori density and Ki-67 labeling index in H. pylori infected children. H. pylori density, Ki-67 labeling index and inflammatory cell infiltration in the p53-positive group were significantly higher than in the p53-negative group. Although intestinal metaplasia was more common in H. pylori infected children (n = 11; 31.4%), there was no difference in the rate of intestinal metaplasia between the p53-positive and p53-negative groups., Conclusions: The present study shows that p53 mutations and higher proliferative activity of glandular epithelium may be related to H. pylori associated gastritis in children. Because p53 mutation does not appear to be associated with intestinal metaplasia, a precursor for gastric cancer in adults, we think that H.pylori associated p53 alterations do not initiate and promote gastric cancer that may occur in adulthood.

Bovine ocular squamous cell carcinoma (OSCC) is the second most common cause of rejection due to neoplasia in slaughterhouses on Sao Miguel Island, Azores, and accounts for significant economic losses. To obtain a better insight into the genesis and neoplastic transformation process of bovine OSCC, abnormal protein expression and proliferation index were assessed by the immunohistochemical evaluation of p53 and Ki67, respectively. OSCC samples were collected from 15 bovines and were classified histologically according to the degree of differentiation into three categories: poorly, moderately, and well differentiated. Immunohistochemistry using polyclonal anti-human p53 antibody and polyclonal anti-human Ki67 antibody was performed. Ten of 15 tumors tested were immunoreactive for p53. Twelve tumors demonstrated Ki67 expression. As in human squamous cell carcinoma, p53 overexpression is frequent in bovine OSCC, providing support for a possible role of the protein in the pathogenesis of this neoplasia. No correlation between the percentage of p53 stained nuclei and the degree of differentiation was observed, although different patterns of staining were seen according to the degree of keratinization of the tumor cells. With the exception of the moderately differentiated OSCC group, Ki67 index showed significant correlation with the histologic pattern, increased proliferation being found in poorly differentiated OSCC (P = 0.013).

The health risks to astronauts exposed to high-LET radiation include possible cognitive deficits. The pathogenesis of radiation-induced cognitive injury is unknown but may involve loss of neural precursor cells from the subgranular zone (SGZ) of the hippocampal dentate gyrus. To address this hypothesis, adult female C57BL/6 mice received whole-body irradiation with a 1 GeV/nucleon iron-particle beam in a single fraction of 0, 1, 2 and 3 Gy. Two months later mice were given BrdU injections to label proliferating cells. Subsequently, hippocampal tissue was assessed using immunohistochemistry for detection of proliferating cells and immature neurons. Routine histopathological methods were used to qualitatively assess tissue/cell morphology in the hippocampal formation and adjacent areas. When compared to controls, irradiated mice showed progressively fewer BrdU-positive cells as a function of dose. This observation was confirmed by Ki-67 immunostaining in the SGZ showing reductions in a dose-dependent fashion. The progeny of the proliferating SGZ cells, i.e. immature neurons, were visualized by doublecortin staining and were significantly reduced by irradiation, with the decreases ranging from 34% after 1 Gy to 71% after 3 Gy. Histopathology showed that in addition to cell changes in the SGZ, (56)Fe particles induced a chronic and diffuse astrocytosis and changes in pyramidal neurons in and around the hippocampal formation. The present data provide the first evidence that high-LET radiation has deleterious effects on cells associated with hippocampal neurogenesis.

The aim of the study was to evaluate the effect of fixation procedures on MIB-1 immunostaining on microwave-treated Papanicolaou-stained slides and to establish protocol for MIB-1 immunostaining on cytological samples without microwave pre-treatment. Cytospins for immunostaining and nuclear suspension for DNA measurement were prepared from human breast cancer cell line (MCF-7). Following fixation, the cytospins were either stained by Papanicolaou, stored in methanol or air-dried. Antigen retrieval by microwave was used before MIB-1 immunostaining only for Papanicolaou-stained cytospins. Air-dried cytospins and cytospins stored in methanol were immunostained without pre-treatment. The percentage of MIB-1 positive cells was compared with the S phase fraction of MCF-7 cells calculated from DNA histograms. Variations in fixation procedures used before Papanicolaou staining had no influence on the percentage of MIB-1 positive cells. The difference between the percentage of the MIB-1-positive cells on microwave-treated Papanicolaou-stained cytospins and on methanol-fixed cytospins without microwave pre-treatment was not significant. There was a strong correlation between the percentage of the MIB-1-positive cells and S phase fraction. Monoclonal antibody MIB-1 recognized Ki-67 antigen in Papanicolaou-stained cytospins treated by microwave as well as in cytospins that were fixed and stored in methanol without microwave pre-treatment.

Recent studies by use of short phosphopeptides showed that forkhead-associated (FHA) domains recognize pTXX(D/I/L) motifs. Solution structures and crystal structures of several different FHA domains and their complexes with short phosphopeptides have been reported by several groups. We now report the solution structure of the FHA domain of human Ki67, a large nuclear protein associated with the cell-cycle. Using fragments of its binding partner hNIFK, we show that Ki67-hNIFK binding involves ca 44 residues without a pTXX(D/I/L) motif. The pThr site of hNIFK recognized by Ki67 FHA is pThr234-Pro235, a motif also recognized by the proline isomerase Pin1. Heteronuclear single quantum coherence (HSQC) NMR was then used to map out the binding surface, and structural analyses were used to identify key binding residues of Ki67 FHA. The results represent the first structural characterization of the complex of an FHA domain with a biologically relevant target protein fragment. Detailed analyses of the results led us to propose that three major factors control the interaction of FHA with its target protein: the pT residue, +1 to +3 residues, and an extended binding surface, and that variation in the three factors is the likely cause of the great diversity in the function and specificity of FHA domains from different sources.

The monoclonal antibody (MAb) Ki-67 is routinely used in clinical studies to estimate the growth fraction of tumors. However, the role of pKi-67, the protein detected by the Ki-67 MAb, remains elusive, although some biochemical data strongly suggest that it might organize chromatin. To better understand the functional organization of pKi-67, we studied its three-dimensional distribution in interphase cells by confocal microscopy and electron tomography. FluoroNanogold, a single probe combining a dense marker with a fluorescent dye, was used to investigate pKi-67 organization at the optical and ultrastructural levels. Observation by confocal microscopy followed by 3D reconstruction showed that pKi-67 forms a shell around the nucleoli. Double labeling experiments revealed that pKi-67 co-localizes with perinucleolar heterochromatin. Electron microscopy studies confirmed this close association and demonstrated that pKi-67 is located neither in the fibrillar nor in the granular components of the nucleolus. Finally, spatial analyses by electron tomography showed that pKi-67 forms cords 250-300 nm in diameter, which are themselves composed of 30-50-nm-thick fibers. These detailed comparative in situ analyses strongly suggest the involvement of pKi-67 in the higher-order organization of perinucleolar chromatin.

Background: The LR domain of marsupial chmadrin is defined by its C-terminal amino acid sequence, which contains several pairs of leucine (L) and arginine (R) residues. The LR domain of chmadrin causes a significant compaction of chromatin over the entire length of chromosomes when it is overproduced. The possible human homologue of chmadrin, Ki-67 antigen (pKi-67), also has a stretch of LR pairs, but with no obvious overall similarity, at its C-terminus., Results: The LR domain of human pKi-67 also induced chromatin compaction, both in human and marsupial cells. A yeast two-hybrid assay and an in vitro binding assay demonstrated that the human LR domain binds to heterochromatin protein 1 (HP1), a well-characterized molecule as a mediator of heterochromatin formation. In fixed cells stained with specific antibodies, the pKi-67 was found to be co-localized partially with HP1 at foci on chromosomes in an early G1 phase. Time-lapse observation in living cells co-expressing the fluorescently tagged proteins showed that the LR domain formed foci on chromosomes over a limited period of the cell cycle from the telophase to early G1 phase and that HP1 subsequently accumulated at these foci of the LR domain., Conclusions: Marsupial chmadrin and human pKi-67 induce chromatin compaction across species, possibly via the interaction of its LR domain with HP1.

In a previous study, we demonstrated that the forkhead associated (FHA) domain of pKi-67 interacts with the novel kinesin-like protein, Hklp2 (Sueishi, M., Takagi, M., and Yoneda, Y. (2000) J. Biol. Chem. 275, 28888-28892). In this study, we report on the identification of a putative RNA-binding protein of 293 residues as another binding partner of the FHA domain of pKi-67 (referred to as NIFK for nucleolar protein interacting with the FHA domain of pKi-67). Human NIFK (hNIFK) interacted with the FHA domain of pKi-67 (Ki-FHA) efficiently in vitro when hNIFK was derived from mitotically arrested cells. In addition, a moiety of hNIFK was co-localized with pKi-67 at the peripheral region of mitotic chromosomes. The hNIFK domain that interacts with Ki-FHA was mapped in the yeast two-hybrid system to a portion encompassed by residues 226-269. In a binding assay utilizing Xenopus egg extracts, it was found that the mitosis-specific environment and two threonine residues within this portion of hNIFK (Thr-234 and Thr-238) were crucial for the efficient interaction of hNIFK and Ki-FHA, suggesting that hNIFK interacts with Ki-FHA in a mitosis-specific and phosphorylation-dependent manner. These findings provide a new clue to our understanding of the cellular function of pKi-67.

Gonadotropin releasing hormone (GnRH) analogs can cause regression of hormone-dependent breast carcinomas via the specific GnRH receptor (GnRH-R). In an attempt to obtain a better understanding of GnRH actions in human breast carcinoma, the expression of GnRH-R was examined immunohistochemically in 58 invasive ductal carcinomas and correlated with various clinicopathological parameters. GnRH-R was immunolocalized in the cytoplasm of carcinoma cells in 37 of 58 invasive ductal carcinoma cases (64%). Immunoreactivity for GnRH-R was also detected focally in the cytoplasm of morphologically normal glandular epithelia adjacent to the carcinoma. A significant correlation was observed between the immunohistochemical expression of GnRH-R and estrogen receptor labeling index (LI; P = 0.030) or progesterone receptor LI (P = 0.0074). There was a significant inverse correlation between GnRH-R immunoreactivity and Ki-67 LI (P = 0.012). No significant correlations were detected between GnRH-R and other clinicopathological parameters, including patient age, menopausal status, stage, tumor size, lymph node status, histological grade and prognosis. This study indicates that GnRH-R is widely distributed in human breast carcinoma cells and regulates GnRH actions locally. Breast carcinomas positive for GnRH-R maintain some hormonal regulatory mechanisms, and GnRH actions may lead to a low proliferative rate in human breast carcinoma.

Quinolones and magnesium deficiency cause similar lesions in joint cartilage of young animals. Chondrocytes cultivated in the presence of quinolones and in Mg-free medium show severe alterations in cytoskeleton and decreased ability to adhere to the culture dish. We investigated whether Mg2+ supplementation can prevent quinolone-mediated effects on chondrocytes in vitro. Chondrocytes cultivated in Dulbecco's modified Eagle's medium/HAM's F-12 medium were treated with ciprofloxacin (80 and 160 microg/ml) and enrofloxacin (100 and 150 microg/ml). Mg2+ was added at a concentration of 0.0612 mg/ml (MgCl) and 0.0488 mg/ml (MgSO4) or a triple dose. In addition, cells were cultivated in Mg-free medium and accordingly treated with Mg2+ supplementation. After 5 days in culture, the number of adherent cells per milliliter was determined. The number of chondrocytes in quinolone-treated groups decreased to 12-36% that of the control group within the culture period. With Mg2+ supplementation, the number of attached cells increased to 40-70% that of control cells. The threefold dose of Mg2+ led to better results than did the single dose. Cell proliferation tested by immunohistochemical staining with Ki67 (clone MIB5) decreased from 70% in control groups to 55%, 48%, and 30% in enrofloxacin-treated groups in a concentration dependent manner (50, 100, and 150 microg/ml). Addition of Mg2+ did not increase the rate of cell proliferation. These results suggest that a great part of quinolone-induced damage is due to magnesium complex formation, as Mg2+ supplementation is able to reduce the effects in vitro. However, quinolone effects on cell proliferation seem to be an independent process that is not influenced by magnesium supplementation.

Background: Medical treatment of benign prostatic hyperplasia (BPH) targets relief of symptoms by causing either relaxation of the prostatic smooth muscle with alpha1 adrenergic blockade, or shrinkage of the gland with 5alpha-reductase inhibitors. We recently demonstrated that alpha1-blockers, such as terazosin, induce apoptosis in prostatic cells. In this study, we examined the combined effect of finasteride and terazosin on the rate of apoptosis and cellular proliferation to investigate their potential synergy at the cellular level., Methods: Prostate specimens were obtained from men who were treated with either finasteride (n = 24), terazosin (n = 42), or combination therapy (n = 10) for varying time periods (1 week to 36 months) for the relief of the symptoms of BPH. The proliferative and apoptotic indices of both stromal and epithelial prostatic cell populations were determined. Antibodies against TGF-beta1 and TbetaRII were used to examine the immunoreactivity of TGF-beta1 and TbetaRII, respectively, in all prostate tissue sections., Results: The apoptotic index in both prostate cell populations was significantly higher following the combination treatment compared to terazosin or finasteride alone. There were no significant changes in the rate of cellular proliferation with any treatment. Furthermore, there was a significant increase in TGF-beta1 expression in the prostates of patients treated with terazosin or combination therapy, while there was no change in TbetaRII expression., Conclusions: These results support the concept that induction of prostate apoptosis is a potential molecular mechanism underlying the combination effect of alpha1 blockade with 5alpha-reductase inhibitors in the effective treatment of BPH. The upregulation of TGF-beta1 implies a role for this ligand as an effector of apoptosis induction in response to alpha1-blockade or finasteride therapy of BPH patients., (Copyright 2001 Wiley-Liss, Inc.)

The Ki-67 antigen (pKi-67) is widely used as a cell proliferation marker protein. Its actual role in the cell cycle progression, however, is presently unclear. Using a two-hybrid screening in yeast, a novel protein, termed Hklp2 (human kinesin-like protein 2), was identified and shown to interact with the forkhead-associated (FHA) domain of pKi-67. Hklp2 has 1388 amino acids and shows a striking similarity (a 53% identity in amino acids) to Xklp2, a plus-end directed kinesin-like motor found in Xenopus. The interaction domain of Hklp2 was mapped to the portion that comprised residues 1017-1237 and that was phosphorylated in vitro by incubating with mitotic but not interphasic HeLa cell extracts. That the interaction was striking in the mitotic extract was also verified. In addition, immunofluorescence using specific antibodies revealed an association between pKi-67 and Hklp2 at the periphery of mitotic chromosomes, largely in close proximity to the centromeres. These findings suggest that pKi-67 is involved in the progression of mitosis via its interaction with Hklp2.

Malignant neoplasms represent one-third of all pediatric salivary gland tumors. Mucoepidermoid carcinoma (MEC) composes 51% of malignant tumors and 16% of all salivary gland neoplasms in pediatrics. Prognostic factors in MEC in pediatric patients have not been well defined. Histopathologic features, clinical outcomes and proliferation markers in 26 pediatric patients (median age 11 years; 19F:7M) with salivary gland MECs were evaluated retrospectively. MEC histocytologic grading used a three-tiered system. Proliferation was assessed by determining the percentage of tumor cells immunoreactive for PCNA and Ki-67. Tumor site was 16 parotid, eight submandibular, one base of tongue and one maxillary lip. Median tumor size was 2.5 cm (range 1.5-5 cm). MEC grade was nine low grade (LG), 15 intermediate grade (IG) and two high grade (HG). Metastatic disease and capsular invasion occurred in five cases, while perineural invasion was noted in three cases. Mean percentage of tumor cells immunoreactive for proliferation markers is as follows: PCNA: LG 9%, IG 17%, HG 32%; and Ki-67: LG 7%, IG 12%, HG 26%. Treatment was surgical in 21 cases, and surgery with chemotherapy and radiotherapy in five cases. Two patients with high grade MECs died of disease (21, 44 months). Twenty-four patients had no evidence of disease at a median follow-up of 104 months (range 30-298 months). MECs were second malignancies in two children with prior radiotherapy and chemotherapy for leukemia and histiocytosis. Low and intermediate grade salivary gland MECS in a pediatric population may have a favorable outcome when compared with high grade MECs. Proliferation markers appear to be linked to histocytologic MEC grade and may provide information regarding biologic behavior of salivary gland MECs in children and adolescents.

Background: Cell proliferation, apoptosis and expression of p53 proteins were studied in human uterine leiomyomas and myometrium during the menstrual cycle and after menopause., Methods: Expression of ki-67 and p53 was analyzed by immunohistochemistry and by immunoblotting. Apoptosis was detected by in situ 3' end labelling of cells with DNA fragmentation., Results: In both the proliferative and the secretory phases, ki-67 expression was higher in leiomyomas than in myometrium and both tissues showed higher expression in the secretory than in the proliferative phase. No difference in apoptotic index was observed between leiomyomas and myometrium or between the proliferative and secretory phases. After menopause, the expression of ki-67 as well as the apoptotic index was lower than in the proliferative and secretory phases and no significant difference between tissues was seen. Both leiomyomas and myometrium showed negative staining for p53. Immunohistochemical results regarding p53 were confirmed by Western blot., Conclusions: Our findings indicate that sex steroids influence the growth of leiomyomas by stimulating cell proliferation rather than by affecting apoptosis. The rate of cell proliferation is higher in fertile age than after menopause and appears to be enhanced under the influence of progesterone.

Although widely used as an operational marker of proliferation, the cell cycle-regulated Ki67 protein is of unknown function. pKi67 is found predominantly in the nucleolus in cycling interphase cells and moves to become perichromosomal during mitosis. We have performed a detailed immunochemical analysis of pKi67 in HeLa cells and report the existence of a novel hyperphosphorylated form in mitosis. Two isoforms can be identified on immunoblots as a consequence of the previously described alternative splicing. In extracts from mitotic cells both these isoforms have considerably reduced mobility. Treatment with phosphatase converts the mitotic form to the interphase form. Immunoprecipitated pKi67 can be phosphorylated in vitro both by cdc2/cyclin B and by protein kinase C, and treatment by PKC leads to the full mobility shift. Treatment of nocodazole-arrested mitotic HeLa cells with staurosporine causes a dephosphorylation of pKi67 to the interphase state and a concomitant change in the localization of pKi67 with movement away from the perichromosomal layer to cytoplasmic dots that colocalize with nucleolin. These data indicate that pKi67 localization is regulated by the action of cell cycle-specific kinase(s) and phosphatase(s). The data presented here provide a starting point for the analysis of pKi67 function and regulation., (Copyright 1999 Academic Press.)

A novel perichromosomal protein, which we have named chmadrin, was identified from rat kangaroo PtK2 cells. The deduced amino acid sequence revealed structural homologies in several limited regions to the Ki-67 antigen (pKi-67). The subcellular localization of chmadrin was found to be similar to that of pKi-67 throughout the cell cycle, that is, predominantly nucleolar during interphase and perichromosomal in the mitotic phase. In addition, a certain population of the protein was found to be localized in heterochromatic foci in interphase nuclei. Transient expression analysis of the truncated proteins corresponding to the conserved regions clearly demonstrated the structural basis for the characteristic cellular localization. Residues 494-778, which show extensive similarity to the corresponding region of pKi-67, were efficiently targeted to nucleoli, whereas a repetitive structure found at the C-terminal portion, whose similarity to pKi-67 is weak, was localized precisely to mitotic chromosomes. The C-terminal portion was designated the 'LR domain' since several LR (leucine and arginine) pairs commonly appear in chmadrin and pKi-67. When overproduced in the interphase nuclei, the LR domain induced the formation of aberrant heterochromatin as a structural constituent. These are the first empirical data suggesting the involvement of perichromosomal proteins in the organization of chromatin structure.

A recent study by Morgan et al. on the mechanism of the heating antigen retrieval (AR) has raised an interesting issue concerning calcium-induced modification of protein conformation demonstrated by immunohistochemistry (IHC). The current study is based on calcium-induced modification of thrombospondin (TSP) and Ki-67, as demonstrated by IHC using seven monoclonal antibodies (MAbs) to TSP and an MAb MIB1. Experiments were carried out on frozen tissue sections of bladder carcinoma and lymph node. Frozen sections were incubated with solutions of 50 mM CaCl2 and/or 10 mM EDTA at 4C overnight before formalin or acetone fixation for TSP and Ki-67, respectively. Sections were then fixed in 10% neutral buffered formalin or acetone before immunostaining. Seven MAbs to TSP, named Ab1 to 7 representing clone numbers of A4.1, D4.6, C6.7, A6.1, B5.2, A2.5, and HB8432, respectively, and MIB1 were utilized as primary antibodies. ABC was used as the detection system and AEC as the chromogen for immunohistochemical staining. An extracellular immunostaining pattern represented a positive result for TSP, and nuclear staining for MIB1. Frozen sections preincubated in 50 mM CaCl2 overnight at 4C showed significant loss of staining and/or altered staining pattern for six of the seven antibodies to TSP and MIB1 compared to positive controls not exposed to CaCl2. Lack of immunostaining of TSP and MIB1 attributable to exposure to CaCl2 could be partially recovered by incubating the frozen sections in EDTA. Calcium-induced modification of protein structure was demonstrated more than 10 years ago on the basis of immunochemical techniques. In this study, similar calcium-induced modification of protein was detectable by IHC in frozen tissue sections, suggesting that calcium-induced modification of protein structure may occur independently of fixation-induced modification. The fact that calcium binding may affect IHC staining is not surprising in view of the fact that antibody/antigen interactions are protein structure-dependent. However, in this experiment the change occurred before and independent of formalin fixation and does not necessarily imply a role for calcium in AR. There may be a valuable role for the use of chemical modification in visualization of protein structure changes in tissue sections by IHC. (J Histochem Cytochem 47:463-469, 1999)

The effect of melanin bleaching on the immunoreactivity of the MIB1-Ki67 antigen in pigmented melanocytic lesions was investigated. Eight paired non-pigmented and heavily pigmented malignant melanomas (6 primary melanomas and 2 secondary melanomas) were selected. Avidin-biotin immunoperoxidase complex (ABC) and microwave antigen retrieval were used in immunostaining. Sections were incubated with 10% H2O2 for 24h before immunostaining with primary antibody MIB1, or after the completion of immunostaining. Non-bleached controls were obtained by conducting the identical staining but omitting the bleaching procedure. In all heavily pigmented lesions bleached by 10% H2O2 before or after immunostaining, the melanin was bleached effectively and MIB1-positively stained cells were clearly seen. Cell counting in the non-pigmented group found that there were no significant differences in the percentage of MIB1-positive melanoma cells (%MIB1) between non-bleached controls and those sections which had been bleached by 10% H2O2 either before or after the immunostaining. The results suggest that hydrogen peroxide can effectively bleach melanin in pigmented melanocytic lesions without significantly affecting MIB1-Ki67 immunolabelling.

To clarify the events leading to the disruption of cell growth control that occurs during the development of pulmonary adenocarcinoma (AC), we used immunohistochemistry to evaluate the expression of G1 cycle regulators, cyclin D1, Rb protein (pRb), and p16 MTS1 protein and the tumour proliferation marker, Ki 67, both in AC of the lung and in its precursor lesion, atypical adenomatous hyperplasia (AAH). The frequency of lesions with cyclin D1 overexpression was relatively high in AAH (47-89%), but was decreased in early AC (28%) and overt AC (35%). The loss of pRb expression was rare in both AAH (0-18%) and early AC (0%), and was infrequent even in overt AC (13%). The loss of p16 expression was also relatively infrequent in both the premalignant and the malignant lesions (11-25%). Our results suggest that overexpression of cyclin D1 is an early event and plays an important part in tumorigenesis in the case of lung AC. However, cyclin D1 overexpression is not required for the development and maintenance of a malignant phenotype. It is likely that some cyclin D1-independent pathways other than Rb and p16 abnormalities have an important role in the malignant transformation from AAH to early AC.