Evaluation of cell proliferation in rat tissues with BrdU, PCNA, Ki-67(MIB-5) immunohistochemistry and in situ hybridization for histone mRNA.

The standard method for assessment of cell proliferation in paraffin-embedded tissue sections is 5-bromodeoxyuridine (BrdU) immunohistochemistry (IHC). BrdU can be administered to laboratory animals via IP injections, is readily incorporated into nuclei during the DNA synthetic phase of the cell cycle, and is detected with an anti-BrdU antibody. This method has several disadvantages, and an accurate method for evaluation of proliferative activity that can substitute for BrdU IHC, when necessary, is of great interest to investigators. Alternative methods for detection of proliferating cells in tissue sections are proliferating cell nuclear antigen (PCNA) IHC, Ki-67 IHC, and in situ hybridization (ISH) for histone mRNA. To determine the optimal choice, we analyzed the correlation of anti-PCNA, anti-Ki-67(MIB-5), and histone mRNA labeling indices (LIs) with anti-BrdU LI in rat highly replicative (renewing) tissues. The correlation between anti-BrdU and histone mRNA LIs, as well as the correlation between anti-BrdU and anti-Ki-67 LIs, was statistically significant. There was no significant correlation between anti-BrdU and anti-PCNA LIs. These results suggest that both ISH for histone mRNA and IHC with MIB-5 are preferable techniques for assessment of cell proliferation in rat paraffin-embedded renewing tissues compared to PCNA IHC. They can substitute for BrdU IHC when necessary.     

Relationship between the RB1 mRNA level and the expression of phosphorylated RB protein in human breast cancers: their relevance in cell proliferation activity and patient clinical outcome

The aim of the present study was to ascertain the relationship between the level of RB1 mRNA and the expression of phosphorylated RB protein and the relevance of these two parameters in cancer cell proliferation and clinical outcome in human breast cancer. Sixty-eight primary human breast cancers were considered. The amount of RB1 mRNA was evaluated by quantitative RT-PCR analysis. The level of RB phosphorylation was immunohistochemical defined by measuring the phosphorylated (pp) RB labelling index (LI). Cell proliferation rate was measured by calculating the Ki67 LI. No relation was found between the RB1 mRNA level and the ppRB LI (p=0.565). Both RB1 mRNA value and ppRB LI were related (in an inverse and direct manner, respectively) to Ki67 LI. RB1 mRNA expression was more strictly associated with KI67 LI (p=0.001) than the ppRB LI (p=0.013). Regarding the patient clinical outcome, the separately considered RB parameters did not reach the prognostic significance. However, patients with low RB1 mRNA quantity and patients with high ppRB LI, taken together, had a significantly shorter disease free and overall survival than the group comprehending patients with high RB1 mRNA value and low ppRB LI, and this despite the low number of patients considered. Our results demonstrated that the ppRB LI was independent of the RB1 mRNA level; that both RB parameters are related to the cell proliferation rate and, if collectively considered, have a high informative value on breast tumour prognosis.     

Histone H3 mRNA in situ hybridization for identifying proliferating cells in human pancreas, with special reference to the ductal system

In general, the incidence of proliferating cells parallels that of carcinogenesis. We have investigated proliferating activity and phenotype expression in epithelial cells in normal tissue, mucinous metaplasia and ductal adenocarcinoma of the pancreas. Twenty-eight resected pancreases (15 cases of pancreatic ductal adenocarcinoma and 13 cases of other diseases) were examined. Formalin-fixed, paraffin-embedded tissue sections were examined for proliferating cell activity using histone H3 mRNA in situ hybridization and immunostaining for Ki-67. In the normal pancreas, the labelling indices for proliferating cells were low and no generating zone was found. The following progressive increase was found in the labelling indices: normal ductal epithelium < mucinous metaplasia without papillary hyperplasia < mucinous metaplasia with papillary hyperplasia < ductal carcinoma. In the pancreatic ductal adenocarcinomas, the S-phase fraction, as defined by the ratio H3-mRNA-labelling index/Ki-67-labelling index, increased as the degree of differentiation decreased. Mucinous metaplasia with papillary hyperplasia showed organoid differentiation toward pyloric mucosa. If used in combination with other proliferative markers on paraffin-embedded tissue sections, histone H3 mRNA in situ hybridization could open broader perspectives on the biology of cell proliferation in the pancreatic ductal system.     

Automatic quantification of immunohistochemically stained cell nuclei based on standard reference cells.

A fully automatic method for quantification of images of immunohistochemically stained cell nuclei by computing area proportions, is presented. Agarose embedded cultured fibroblasts were fixed, paraffin embedded and sectioned at 4 microm. They were then stained together with 4 microm sections of the test specimen obtained from bladder cancer material. A colour based classifier is automatically computed from the control cells. The method was tested on formalin fixed paraffin embedded tissue section material, stained with monoclonal antibodies against the Ki67 antigen and cyclin A protein. Ki67 staining results in a detailed nuclear texture with pronounced nucleoli and cyclin A staining is obtained in a more homogeneously distributed pattern. However, different staining patterns did not seem to influence labelling index quantification, and the sensitivity to variations in light conditions and choice of areas within the control population was low. Thus, the technique represents a robust and reproducible quantification method. In tests measuring proportions of stained area an average standard deviation of about 1.5% for the same field was achieved when classified with classifiers created from different control samples.     

The Prognostic Value of Mitotic Activity Index (MAI), Phosphohistone H3 (PPH3), Cyclin B1, Cyclin A,and Ki67, Alone and in Combinations,in Node-Negative Premenopausal Breast Cancer

Proliferation, either as the main common denominator in genetic profiles, or in the form of single factors such as Ki67, is recommended for clinical use especially in estrogen receptor-positive (ER) patients. However, due to high costs of genetic profiles and lack of reproducibility for Ki67, studies on other proliferation factors are warranted. The aim of the present study was to evaluate the prognostic value of the proliferation factors mitotic activity index (MAI), phosphohistone H3 (PPH3), cyclin B1, cyclin A and Ki67, alone and in combinations. In 222 consecutive premenopausal node-negative breast cancer patients (87% without adjuvant medical treatment), MAI was assessed on whole tissue sections (predefined cut-off ≥10 mitoses), and PPH3, cyclin B1, cyclin A, and Ki67 on tissue microarray (predefined cut-offs 7th decile). In univariable analysis (high versus low) the strongest prognostic proliferation factor for 10-year distant disease-free survival was MAI (Hazard Ratio (HR)=3.3, 95% Confidence Interval (CI): 1.8-6.1), followed by PPH3, cyclin A, Ki67, and cyclin B1. A combination variable, with patients with MAI and/or cyclin A high defined as high-risk, had even stronger prognostic value (HR=4.2, 95%CI: 2.2-7). When stratifying for ER-status, MAI was a significant prognostic factor in ER-positive patients only (HR=7.0, 95%CI: 3.1-16). Stratified for histological grade, MAI added prognostic value in grade 2 (HR=7.2, 95%CI: 3.1-38) and grade 1 patients. In multivariable analysis including HER2, age, adjuvant medical treatment, ER, and one proliferation factor at a time, only MAI (HR=2.7, 95%CI: 1.1-6.7), and cyclin A (HR=2.7, 95%CI: 1.2-6.0) remained independently prognostic. In conclusion this study confirms the strong prognostic value of all proliferation factors, especially MAI and cyclin A, in all patients, and more specifically in ER-positive patients, and patients with histological grade 2 and 1. Additionally, by combining two proliferation factors, an even stronger prognostic value may be found.     

Relation of proliferative activity to survival in patients with advanced gastric cancer

The proliferative activities of 98 biopsied specimens of advanced gastric cancers were measured by the in vitro bromodeoxyuridine (BrdU) labelling method. BrdU labelling indices (LIs) varied from 4.8 to 45.1%. There was no correlation of BrdU LIs and histological type, distant metastasis, serosal invasion, macroscopic type or tumor size. However, BrdU LIs of tumors with lymph node metastases were significantly higher than those without them. Patients with tumors showing high BrdU LIs had a three-fold relative risk of death, as compared with those showing low BrdU LIs. When the BRdU LIs and all the clinicopathological parameters were entered simultaneously into the Cox model, BrdU LIs, peritoneal dissemination and serosal invasion emerged as independent prognostic parameters. These results indicate that the in vitro BrdU labelling method using biopsied materials may be useful in predicting lymph node metastasis preoperatively and designing operative procedures for individual patients.     

Prognostic implication of CDC25A and cyclin E expression on primary breast cancer patients

Defect in cell cycle control is a hallmark character of cancer. We have investigated the association of Ki67 labeling index, cyclin E and CDC25A expressions with clinical follow-up data in breast carcinomas. Flow cytometry was used to detect gene amplification of cyclins in 44 tumor tissue with invasive breast carcinomas. Multivariate Cox proportional hazard ratio test was used to show the correlations. Cyclin E or CDC25A were upregulated in 34% of the tumors. Among the whole total material, expression of cyclin E and of CDC25A were found upregulated in 31.9% and 39.4% of cells, respectively. Both CDC25A and cyclin E protein expression levels were correlated with Ki67 expression level (p < 0.001). In addition, the expression of CDC25A was associated significantly with poor survival (P = 0.028), whereas no correlation was found with cyclin E. These findings suggest a possible prognostic value for CDC25A as a cell cycle marker and may imply in characteristic of high risk breast cancer patients.     

Correlation between the expression of cyclin A protein and p53 activity in oral squamous cell carcinomas

Cyclins and wild-type p53 protein are prime cell cycle regulators and may be involved in tumorigenesis. Cyclin A is a late S cyclin and its abnormalities have been reported in several cancers, including oral squamous cell carcinomas. To explore whether aberrant G1/S in p53 mutant tumours leads to increased cyclin A protein in oral squamous cell carcinomas (OSCC), a total of 39 samples were evaluated for the expression of cyclin A and p53 protein by an immunohistochemical method using a labelled polymer assay. These samples comprised two hyperkeratotic and three oral premalignant lesions (two moderate and one severe dysplastic lesions), and 27 OSCC, together with seven healthy controls. The results demonstrated that the cyclin A protein was localized and highly expressed in the nuclei of the tumour cells. Although there was no correlation between cyclin A detection and the local lymph node involvement, a positive correlation was noted between the positivity of cyclin A and p53 protein (p <0.05). The results suggested that cyclin A may contribute to the progression of oral cancer and correlated to some degree with that of the p53 gene activity.     

Differential role of D cyclins in the regulation of cell cycle by influencing Ki67 expression in HaCaT cells

D-type cyclins are important regulatory proteins of the G1/S phase of the cell cycle however, their specific functions are only partially understood. We show that silencing of individual D-type cyclins has no effect on the proliferation and morphology of Immortalized non-tumorigenic human epidermal (HaCaT) cells, while double and triple D cyclin silencing results in the failure of the cytokinesis leading to the appearance of large multinucleated cells. Both CDC20 and Ki67 mRNA is downregulated in these cells. Ki67 mRNA silenced cells show similar multinucleated cellular phenotype as double or triple D cyclin silenced cells without affecting D cyclin expression, suggesting that Ki67 is necessary for normal G2/M transition. Our data have revealed that cyclin D1 may have a leading role in G1/S phase regulation and suggest an incomplete functional overlap among D cyclins. Our results indicate that beside their well-known functions during the G0-G1/S phase, D-type cyclins play a pivotal role in the regulation of mitosis via influencing Ki67 expression in a downstream manner probably through E2F1 activation in HaCaT cells.     

Proliferation in murine epidermis after minor mechanical stimulation. Part 1. Sustained increase in keratinocyte production and migration

It was our objective to obtain an insight into the details and dynamics of the cell proliferative changes following minor barrier disruption, the mechanisms of recovery, and their regulation. Hair of the dorsal area of DBA2-mice was removed and the epidermis was tape stripped. Tritiated thymidine was injected into groups of mice at daily intervals thereafter. Labelling and nuclear densities were measured at several time intervals later in the various epidermal strata to characterize cell production and cell fluxes through the tissue. A dramatic proliferative response was observed at 24 h when the labelling density increased more than sixfold in the basal layer. Labelled cells rapidly appeared in suprabasal layers within a few hours in large quantities while this process took over 2 days in normal skin. Some cycling cells were also found in the suprabasal layer (pulse labelling at 24 h) in contrast with the controls. The cellular flux through the suprabasal layers was drastically (20-fold) increased and the transit time was shortened. Although the nuclear density in the basal layer showed only moderate changes it increased four-fold in the suprabasal layer within 5 days. A kinetic model analysis suggested that the cell cycle time of proliferative cells dropped from a normal value of about 200 h to less than 12 h post tape strip. After 7 days, the proliferative activation still persisted, even though at 3 days post tape strip the stratum corneum had been re-established. Hence, a mild mechanical alteration with removal of some parts of the cornified layer in mouse backskin epidermis triggers a huge proliferative response with massive overproduction of cells that lasts at least 7 days. Our findings suggest that the re-establishment of the cornified layer does not immediately shut down cell proliferation and that more complex, slower (long-term) regulatory processes are involved.     

FGF-16 is released from neonatal cardiac myocytes and alters growth-related signaling: a possible role in postnatal development

FGF-16 has been reported to be preferentially expressed in the adult rat heart. We have investigated the expression of FGF-16 in the perinatal and postnatal heart and its functional significance in neonatal rat cardiac myocytes. FGF-16 mRNA accumulation was observed by quantitative RT-PCR between neonatal days 1 and 7, with this increased expression persisting into adulthood. FGF-2 has been shown to increase neonatal rat cardiac myocyte proliferative potential via PKC activation. Gene array analysis revealed that FGF-16 inhibited the upregulation by FGF-2 of cell cycle promoting genes including cyclin F and Ki67. Furthermore, the CDK4/6 inhibitor gene Arf/INK4A was upregulated with the combination of FGF-16 and FGF-2 but not with either factor on its own. The effect on Ki67 was validated by protein immunodetection, which also showed that FGF-16 significantly decreased FGF-2-induced Ki67 labeling of cardiac myocytes, although it alone had no effect on Ki67 labeling. Inhibition of p38 MAPK potentiated cardiac myocyte proliferation induced by FGF-2 but did not alter the inhibitory action of FGF-16. Receptor binding assay showed that FGF-16 can compete with FGF-2 for binding sites including FGF receptor 1. FGF-16 had no effect on activated p38, ERK1/2, or JNK/SAPK after FGF-2 treatment. However, FGF-16 inhibited PKC-alpha and PKC-epsilon activation induced by FGF-2 and, importantly, IGF-1. Collectively, these data suggest that expression and release of FGF-16 in the neonatal myocardium interfere with cardiac myocyte proliferative potential by altering the local signaling environment via modulation of PKC activation and cell cycle-related gene expression.     

Controversial relationship between the expression of the RB pathway components and RB protein phosphorylation in human breast cancer

Recent data challenge the relevance of the RB pathway to cancer based on RB inactivation, at least in breast tumors. To obtain information on the actual role of the components of the RB pathway in tumor progression we decided to investigate whether their quantitative changes were associated with variations in the level of RB phosphorylation in human breast cancer. A series of 68 human primary breast carcinomas was studied. Five cases were excluded from the study due to their lack of RB expression. In the remaining 63 cases the expression of cyclin D1, cdk4, cyclin E, and INK4a mRNA was assessed by real-time RT-PCR. The level of RB phosphorylated protein (ppRB) and p27 expression was immunohistochemically analyzed by measuring the percentage of stained cells (labeling index, LI). Cell proliferation rate was measured by Ki67 LI evaluation. The ppRB LI ranged from 5.2 to 73.8 and, as expected, was strongly related to the Ki67 LI (r=0.80; p<0.001). The expression of cyclin D1 mRNA, expressed in arbitrary units (a. u.), ranged from 1.15 to 123.0 and was inversely related to the ppRB LI (p=0.021) and Ki67 LI (p<0.001). Neither the cdk4 (range from 0.07 to 1.13 a. u.) nor the cyclin E (range from 0.13 to 9.27 a. u.) mRNA expression was significantly associated with the ppRB LI (p=0.962 and p=0.103, respectively). Cyclin E was related to Ki67 LI (p=0.022). Both INK4a mRNA (range from 0.01 to 0.60 a. u.) and p27 (LI from 0.0 to 73.1) values were inversely related to the ppRB LI (p=0.022 and p=0.014, respectively). Cyclin D1, cdk4, and cyclin E mRNA expressions were not significantly related to one another. In human primary breast cancers, the expression levels of the factors known to facilitate the cell cycle progression by RB protein phosphorylation were not positively related to ppRB-LI. Pathological increases of cyclin D, cdk4, and cyclin E are very likely associated with other biological functions other than their well-established action on cell cycle progression.     

Immunohistochemical expression of the p53, mdm2, p21/Waf-1, Rb, p16, Ki67, cyclin D1, cyclin A and cyclin B1 proteins and apoptotic index in T-cell lymphomas

Fifty-seven cases of T-cell lymphomas (TCL) including 5 lymphoblastic (T-LBL) and 52 peripheral TCL (PTCL) were analyzed by immunohistochemistry for the expression of p53, mdm2, p21, Rb, cyclin D1, cyclin A, cyclin B1, and Ki67/MIB1 proteins and 39/52 PTCL were also analyzed for the expression of p16 protein and for the presence of apoptotic cells by the TUNEL method. The aim was to search for abnormal immunoprofiles of p53 and Rb growth control pathways and to determine the proliferative activity and the apoptotic index of TCL. Abnormal overexpression of p53, p21 and mdm2, in comparison to normal lymph nodes, was found in 12/57, 10/57 and 2/57 cases of TCL, respectively. Abnormal loss of Rb and p16 expression was found in 1/57 and 2/39 cases, respectively, whereas abnormal overexpression of cyclin D1 was not detected in any of the 57 cases. Our data revealed entity-related p53/p21/mdm2 phenotypes. Indeed, most nodal and cutaneous CD30+ anaplastic large cell lymphomas (ALCL) showed concomitant overexpression of p53 and p21 proteins (7/8 cases), and mdm2 was overexpressed in 2 p53-positive nodal ALCL. In contrast, overexpression of p53 was found in 3/17 cases of nodal peripheral TCL unspecified (PTCL-UC) and 2/7 non-ALCL cutaneous pleomorphic TCL. Overexpression of p21 protein was detected in 2/3 p53-positive PTCL-UC and in 1/2 p53-positive non-ALCL cutaneous pleomorphic TCL. Finally, all the remaining 25 cases of TCL did not show p53 and p21 overexpression. Overall, the p53+/p21+ phenotype in 10/57 TCL suggests wild-type p53 capable of inducing p21 expression. The highest apoptotic index (AI) was found in ALCL and a positive correlation between apoptotic index and Ki67 index (p<0.001) was detected. Ki67, cyclin A and cyclin B1 expression was found in all 57 TCL and on the basis of the combined use of these 3 variables, 3 groups of proliferative activity could be determined: a) high in ALCL and T-LBL, b) low in mycosis fungoides (MF) and gammadelta hepatosplenic TCL, and c) intermediate in the remaining TCL entities. The proliferative activity in the 12 p53 overexpressing cases was higher in comparison to the 45 p53-negative cases. Ki67 expresion in more than 25% of tumour cells showed significant correlation with p53 overexpression (p<0.001). Rb expression tended to be parallel to Ki67, cyclin A and cyclin B1 expression in all but one case of nodal PTCL-UC which displayed loss of RB expression. Interestingly, this case was p53-negative, whereas the p53-positive cases were Rb-positive. These findings suggest that different pathogenetic routes may function in some TCL, involving either the p53 or, less frequently, the Rb pathways.     

Expression of proliferation marker Ki67 correlates to occurrence of metastasis and prognosis, histological subtypes and HPV DNA detection in penile carcinomas

Clinical outcome of penile squamous cell carcinoma (PSCC) largely depends on the presence of lymph node metastasis. In search of a valuable marker predicting the risk for metastasis, the expression of Ki67 was investigated immunohistochemically in primary tumors and compared to presence of inguinal lymph node metastasis. As human papilloma virus (HPV) is thought to affect Ki67 expression, we evaluated whether occurrence of HPV DNA correlates to Ki67 score or metastatic potential. Samples originated from patients subjected to resection of invasive SCC of penis. Immunohistochemistry was done on paraffin-embedded sections using a monoclonal antibody against Ki67. After DNA isolation from paraffin embedded tissue the presence of HPV 6/11, HPV 16 and HPV 18 DNA was analyzed by PCR. Statistical analysis was done using two tail unpaired t test and Chi-square test. Four of 28 patients showed a weak Ki67 expression, without displaying lymph node metastasis. Among 17 patients showing an intermediate Ki67 index, eight exhibited metastases while in all seven patients with a strong expression of Ki67 lymph node metastases were found. The median Ki67 expression in metastastic lesions was significantly different (50.3%) from tumors without lymph node metastasis (31.8%) (p=0.024). Furthermore, a correlation between presence of HPV DNA and strong Ki67 expression was determined (p=0.009). Since our study demonstrated a strong Ki67 labeling index significantly associated to positive lymph nodes, we suggest Ki67 expression as a prognostic marker for lymph node metastasis in penile squamous carcinoma.     

CK8 correlates with malignancy in leukoplakia and carcinomas of the head and neck

Screening of head and neck carcinoma patients with the proteomics-based AMIDA technology yielded a set of tumour-associated antigens, including the intermediate filament protein cytokeratin 8 (CK8). The expression pattern and specificity of CK8 was compared with those of the established markers pan-cytokeratins and CK13, and with that of the proliferation marker Ki67. Expression of CK8 correlated positively with malignancies of the head and neck areas. CK8 was not expressed in healthy epithelium, except for some rare cases of cells of the basal layer and laryngeal tissue. In contrast, the vast majority of head and neck squamous cell carcinomas and metastases strongly expressed CK8. Interestingly, CK8 de novo expression correlated with dysplastic areas of oral leukoplakic lesions, while hyperplastic leukoplakia remained CK8-negative but strongly panCK and CK13 positive. Thus, CK8 is an attractive marker molecule for a differentiated diagnosis of leukoplakia and head and neck carcinomas, which possesses notedly improved specificity as compared with panCK and CK13.     

Location of cell cycle regulators cyclin B1, cyclin A, PCNA, Ki67 and cell cycle inhibitors p21, p27 and p57 in human first trimester placenta and deciduas

Although placental development and implantation depend on the coordination of trophoblast proliferation, differentiation and invasion, little is known about the cell cycle regulators that govern the control of these events. The hypothesis that the coordinated expression of cell cycle progression and inhibition factors will determine whether cytotrophoblasts proliferate or undergo cell cycle arrest or cell cycle exit allowing subsequent differentiation was tested. The cell cycle promotors cyclin A, cyclin B1, PCNA, Ki67 and the cell cycle inhibitors p21, p27 and p57 were immunolocalized in tissue sections of first trimester pregnancies (weeks 6 and 9–12). Double staining with cytokeratin 7 allowed unambiguous identification of extravillous cytotrophoblast (EVT) in the decidua. Villous cytotrophoblasts were immunolabelled for Ki67 and cyclin A but only few were stained with anti-cyclin B1. The syncytiotrophoblast was devoid of immunoreactivity for any of the cell cycle progression factors. It expressed especially p21, whereas p27 and p57 were predominantly found in villous cytotrophoblasts. PCNA, Ki67, cyclin A and cyclin B1 were immunolocalized in proximal and distal EVTs of anchoring villi and in EVT which had invaded the upper decidual segments. All EVTs strongly expressed p27 and p57, but not p21. These data clearly suggest different functions for p21, p27 and p57 in placental development with distinct roles for p21 and p57 in syncytiotrophoblast and EVT differentiation, respectively. p27 appears to be involved in both the processes. The results may also challenge the concept of differential mitotic activity in the proximal and distal parts of the first trimester cytotrophoblast cell column, but more functional studies are clearly needed. The presence of p27 and p57 in EVT cells, which invade the deciduas deeply, may account for the loss of mitogenic potential of these cells.     

Simultaneous quantification of nucleoproteins and comparison of methyl green-pyronin Y and Feulgen staining in sections of oral squamous cell carcinoma,dysplastic lesions and normal mucosa

A fundamental difference between normal cells and tumor cells is the proliferative activity of the nucleus and nucleolus, which increases progressively from normal to oral dysplastic mucosa to oral squamous cell carcinoma (OSCC). This activity is evaluated routinely using hematoxylin and eosin (H & E) staining, but in some cases, inter-observer variability occurs among pathologists. We evaluated cellular proliferation by staining sections with the methyl green-pyronin Y procedure and the Feulgen reaction. We also compared the efficacy of methyl green-pyronin Y and Feulgen staining for studying nuclear and nucleolar features in oral dysplastic mucosa and in different grades of OSCC. Sections cut from formalin fixed, paraffin embedded blocks of five normal mucosa, 15 dysplastic mucosa, 10 well-differentiated OSCC, 10 moderately differentiated OSCC and five poorly differentiated OSCC cases were stained with Hematoxylin and Eosin, methyl green-pyronin Y and the Feulgen reaction. The mean diameters of the nuclei and number of nucleoli showed significant differences. A progressive increase in diameter of the nucleus and number of nucleoli was observed from normal mucosa through poorly differentiated OSCC. We observed that methyl green-pyronin Y stain is more useful than Feulgen and hematoxylin and eosin for simultaneous quantitative assessment of both RNA and DNA. The simplicity of this technique makes it a valuable tool even for daily routine examination.     

Immunohistochemical distribution of cell cycle proteins p27, p57, cyclin D3, PCNA and Ki67 in normal and diabetic human placentas

The placenta is a regulator organ for many metabolic activities between mother and fetus. Therefore, fetal growth is directly related to the placental development. Placental development is a series of events that depend on the coordinated action of trophoblasts’ proliferation, differentiation and invasion. Studies on cell cycle related proteins which control these events are fairly limited. How placental tissue proliferation is affected by diabetes is not exactly known yet. Therefore in this study, the immunohistochemical localizations of cell cycle related proteins like PCNA, Ki67, cyclin D3, p27 and p57 in the differentiation, proliferation and apoptosis mechanisms of normal and diabetic placentas were investigated. Information on cell cycle related proteins that control these events is limited and how they are affected in diabetes mellitus is not fully understood yet. Therefore, in this study, to understand the role of cell cycle regulators in diabetic placentas we aimed to determine the spatio-temporal immunolocalizations of cell cycle regulators in diabetic and normal human term placentas. Term placentas were obtained from diabetic women and from normal pregnancies with informed consent following caesarean deliveries. Placental samples were stained via immunohistochemistry with PCNA, Ki67, cyclin D3, p27 and p57 antibodies and were examined by light microscopy. When compared to control placentas, PCNA, Ki67 and cyclin D3 staining intensities significantly increased in villous parts of diabetes group. Moreover, Ki67 and cyclin D3 stainings also significantly increased in basal plates and chorionic plate respectively. In chorionic plates, p27 and p57 staining intensities significantly decreased in diabetic group. p57 staining also significantly decreased in villous parts of diabetic placentas. Placental abnormalities seen in diabetic placentas could be associated with proliferation and cell cycle arrest mechanisms’ alterations occurred in diabetes mellitus.     

PCNA、Ki67 在胰腺疾病中的研究进展

PCNA、Ki-67是与细胞增殖有关的核抗原,在增殖的组织细胞中呈阳性表达,反映组织细胞的增殖活性,是细胞增殖的重要标记物。PCNA、Ki-67在正常发育的胚胎组织、糖尿病、胰腺肿瘤、胰岛移植等胰腺疾病及其他疾病中均高表达,同时也与其他系统肿瘤和疾病密切相关。PCNA、Ki-67作为增殖指标可以用于评价胰腺疾病、胰岛细胞移植后细胞再生数量及其他疾病的诊断、治疗及判断预后。目前已将它们视为细胞的标志物,用于细胞增殖的动力学研究,在临床病理上具有很大的应用前景。未来PCNA、Ki67将广泛应用于临床及基础研究,尤其用于研究胰腺疾病的新靶点、探索糖尿病的发病机制,对疾病的预防和治疗及胰岛移植具有一定的应用前景及意义。     

Modalities of synthesis of Ki67 antigen during the stimulation of lymphocytes

The antibody Ki67 is currently used to evaluate the proliferative fraction of solid tumors and some hematological malignancies. We have used phytohemagglutinin (PHA)-stimulated peripheral blood lymphocytes as a model to study the entry of quiescent cells into cell cycle and to follow their progress to the next cycle. Flow cytometric analysis of lymphocyte samples stained with the antibody Ki67 and a DNA marker has allowed us to follow the expression of Ki67 antigen (Ki67 Ag) as a function of the position of the cells in the cell cycle. The use of drugs blocking the stimulated lymphocytes in different phases of the cell cycle permitted us to demonstrate that Ki67 Ag expression started from the beginning of the first S phase. The level of Ki67 Ag increased during S phase until mitosis, when its expression was maximal. After division, the cells in G1 phase showed a decrease in Ki67 Ag expression (possibly corresponding to degradation) until they reentered S phase, when the level of Ki67 Ag increased again. The results confirm that the expression of Ki67 Ag is related to the proliferative state of the cells and suggest that it may be used to determine the proliferative cell fraction in hematopoietic tissues.