Histone H3 messenger RNA in situ hybridization for identifying proliferating cells in formalin-fixed rat gastric mucosa

To devise a more sensitive method for identifying proliferative cells in routinely formalin-fixed, paraffin-embedded tissues, we applied an in situ hybridization (ISH) technique for the detection of histone H3 mRNA in rat gastric mucosa and amplified the signal by a silver intensification method. ISH was performed using a Fluorescein-labelled, single-stranded DNA probe for the human histone H3 gene. To determine the optimal conditions for detecting H3 mRNA in rat gastric mucosa, we tested the effect of changing conditions, such as fixation time and digestion time, by a proteinase before hybridization. Next, the proliferation indices obtained using H3 ISH were compared with those obtained using bromodeoxyuridine (BrdU) immunohistochemistry. In normal rat gastric mucosa, H3 ISH- and BrdU-positive cells were confined to the neck region of both fundic and pyloric mucosa. The two labelling indices were almost the same. In all the serial sections studied, H3 ISH-positive cells were almost always BrdU-positive too. Taken together, these results indicate that the H3 ISH technique is useful for the evaluation of proliferative activity in gastric epithelial cells by virtue of its detection of S-phase cells.     

Mapping of the binding of platelet-derived growth factor to distinct domains of the basement membrane proteins BM-40 and perlecan and distinction from the BM-40 collagen-binding epitope

Iron is required for cell proliferation of all living species. Moreover, iron excess may be involved in the development of hepatocellular carcinoma. In this study we analyzed the effects of deferoxamine, an iron chelator, on normal porcine hepatocyte proliferation. We confirmed that hepatocytes isolated from young pigs proliferate in the presence of insulin and fetal calf serum as shown by [3H] methyl-thymidine incorporation, presence of mitotic figures and increase in cell number. This was paralleled by nuclear expression of p34cdc2 and its associated histone H1 kinase activity. In the presence of deferoxamine, [3H] methyl-thymidine incorporation, expression of nuclear proteins (p34cdc2 and PCNA) and H1 kinase activity were drastically reduced. In addition, in contrast with control cultures, cells in S-phase were not detected by flow cytometry. These data suggest that iron chelation by deferoxamine can arrest the progression of porcine hepatocytes in the G1 phase of the cell cycle.     

Effects of incubation temperature on the energetics of embryonic development and hatchling morphology in the Brisbane river turtle Emydura signata

AIMS: To investigate the malignant potential of lichen sclerosus, a study using the cell proliferation marker Ki67 comparing lichen sclerosus with and without associated squamous cell carcinoma was performed. METHODS AND RESULTS: Formalin-fixed, paraffin-embedded slides of 13 cases of lichen sclerosus with associated carcinoma, and 31 cases without associated carcinoma, including 16 random cases, seven with epidermal thickening and eight with epidermal thinning, were examined by the immunoperoxidase technique for Ki67, a cell proliferation marker. Ki67 reactivity was mostly seen in the basal and parabasal cells in both groups of lichen sclerosus and this pattern was similar to normal skin, squamous cell hyperplasia and analogous to that of one form of squamous cell carcinoma. There was a mean of 50 Ki67 positive cells per 100 basal cells in lichen sclerosus with associated squamous cell carcinoma; however, in squamous cell hyperplasia adjacent to carcinoma this rose to 90 Ki67 positive cells per 100 basal cells. In lichen sclerosus without associated carcinoma, the random cases had a count of 53 per 100 basal cells, those with epidermal thickening 53 and those with thinning 42. Non-genital normal skin had a count of 71 per 100 basal cells. CONCLUSION: The lack of qualitative differences of Ki67 expression in normal skin, in lichen sclerosus with and without carcinoma, in squamous cell hyperplasia and in one form of squamous cell carcinoma indicates that these conditions share a common localized pattern of cell proliferation and does not support or deny the malignant potential of lichen sclerosus. The higher Ki67 count in squamous cell hyperplasia adjacent to carcinoma could indicate premalignancy or a reaction to the carcinoma. In patients without carcinoma, the higher Ki67 count in thickened lichen sclerosus compared to thinned suggests that some or all of the cases of thickened lichen sclerosus were lichen sclerosus with squamous cell hyperplasia or that lichen simplex chronicus superimposed on lichen sclerosus has a higher Ki67 expression or that the distinction between squamous cell hyperplasia and lichen simplex chronicus is only one of terminology.     

Labeling of cycling corneal endothelial cells during healing with a monoclonal antibody to the Ki67 antigen (MIB-1)

PURPOSE: To assess the efficacy of labeling actively cycling corneal endothelial cells by using a monoclonal antibody to the Ki67 antigen (MIB-1) and to determine what changes in f-actin and ZO-1 organization are associated with entry into the cell cycle during wound healing under different culture conditions. METHODS: Three corneal buttons (6 mm diameter) were punched from each cornea of 15 cats. After a mechanical scrape injury (2 mm diameter) was made, buttons were cultured for 24, 48, or 72 h in serum-free media (SFM), SFM plus 10% fetal calf serum, or SFM plus basic fibroblast growth factor (bFGF). Buttons were single and double labeled by using phalloidin, anti-ZO-1, and MIB-1. Counts of Ki67-positive cells were used to determine the number of actively cycling endothelial cells. RESULTS: After culture in SFM, wounds healed by cell spreading with maintenance of normal apical f-actin and ZO-1 organization; Ki67-positive cells were detected near the leading edge in some areas. A significant increase in the number of cycling cells was measured after 48 h of culture in bFGF as compared with SFM (p<0.05); serum increased the number of cycling cells more than both SFM and bFGF (p<0.05). In all cases, positive MIB-1 staining was not observed until 48 h after injury, was limited to cells actively spreading over the wound area, and was diminished after wound closure (72 h). Double labeling demonstrated that endothelial cells exhibited a fibroblastic phenotype in some central areas of cell proliferation after culture in serum or bFGF, but, in general, apical cell border-associated f-actin and ZO-1 organization was partially maintained in most Ki67-positive cells. CONCLUSION: The data suggest that spreading corneal endothelial cells are capable of proliferating and can respond to growth factors, but that dedifferentiation or fibroblastic transformation is not required before entry into the cell cycle. Overall, the MIB-1 antibody appears to be ideally suited to the study of corneal endothelial proliferation during wound healing.     

Regional differences in bromodeoxyuridine uptake, expression of Ki-67 protein, and nucleolar organizer region counts in glioblastoma multiforme

To investigate intratumoral differences in indices of tumor cell proliferation, we measured the bromodeoxyuridine labeling index (BrdUrd LI), the Ki-67 protein proliferating cell indices (PCIs) determined by monoclonal antibody MIB 1 in microwave-processed paraffin sections (MIB 1 PCI) and in some cases by monoclonal antibody in frozen sections (Ki-67 PCI), and counts of argyrophilic nucleolar organizer regions (AgNORs) in 20 glioblastomas. In the most actively proliferating areas, MIB 1 and Ki-67 PCIs correlated well with the BrdUrd LI and with each other, while AgNOR counts correlated less strongly with these indices. In less active areas, the MIB 1 PCI and BrdUrd LI changed concomitantly from one area to another within a tumor except in areas of pseudopalisading with necrosis; in these areas the BrdUrd LI decreased significantly compared with neighboring tumor tissue, while the MIB 1 PCI did not. There was very little staining of gemistocytic nuclei with either anti-BrdUrd or MIB 1 monoclonal antibodies; this supports the concept that gemistocytes are mainly quiescent cells. AgNORs in all of the above-mentioned areas varied from tumor to tumor, which suggests that they may indicate some cellular activity other than proliferation. The close correlation between the BrdUrd LI and Ki-67 protein PCIs in corresponding regions of glioblastomas suggests that MIB 1 staining of microwave-processed paraffin sections can be used to evaluate the growth potential of individual glioblastomas and possibly of other gliomas as well.     

Measurement of DNA content and of tritiated thymidine and bromodeoxyuridine incorporation by the same cells

We tested a method of measuring DNA content (Feulgen) and tritiated thymidine ([3H]-T) and bromodeoxyuridine (BrdU) incorporation by the same cell. Initial experiments showed that Feulgen hydrolysis denatured the DNA of fixed cells sufficiently to allow detection of incorporated BrdU with monoclonal antibodies. MCa-11 cells were then double-labeled with [3H]-T and BrdU, placed on slides, and Feulgen stained. Next, absorption cytometry was performed to measure the DNA content of randomly selected cells. Feulgen staining and the development and removal of either the [3H]-T or the BrdU grains after DNA measurements did not interfere with subsequent detection of the grains from the other label, and BrdU and [3H]-T can be used reliably in combination for identification of S-phase cells. This method may eventually allow the use of microscope-based image analysis to selectively measure the DNA contents and the BrdU/[3H]-T labeling of non-transformed stromal and cancer cells in solid tumors, thereby providing new insights into the growth kinetics of these heterogeneous cell populations.     

Measurement of proliferation activities in human tumor models: a comparison of flow cytometric methods

Proliferating cells in tumors may be of considerable relevance in cancer therapy. Not only do such cells dictate the rate of tumor progression, but evidence exists that they may also play an important role in the diagnosis and prognosis of tumor regrowth. Consequently, the identification of this subset of cells in the overall neoplastic cell population is of considerable importance. The aim of the present investigations was to compare four flow cytometric methodologies commonly used to study cell proliferation. These included nuclear antigen Ki67 detection, acridine orange (AO) and bromodeoxyuridine (BrdUrd) staining, and percent S-phase determinations. Three human tumor cell lines (HEp3, A549, H226) were examined in various stages of growth. Further, a direct comparison was made of the proliferation activities of HEp3 cells grown in culture or as xenografts in nude mice. The results showed that of the techniques investigated, detection of the nuclear antigen Ki67 may be most useful for marking proliferating tumor cells and determining tumor growth fractions.     

Structural features in the 3'' external transcribed spacer affecting intragenic processing of yeast rRNA

A total of 62 patients of gastric carcinoma were studied to find a correlation between newer prognostic indicators like cell proliferative indices including Nucleolar Organizer regions (AgNORs), Ki 67 Labelling Index and Epidermal Growth Factor Receptor (EGFR) expression with the various, histopathological criteria and compared with 30 controls of non neoplastic gastric diseases. EGFR expression was positive in 48(77.4%) cases. The Ki 67 labelling indices ranged from 0 to 50% with a mean of 21.35 +/- 17.88% among the cases. AgNOR counts ranged from 1.64 to 4.49 with a mean of 3.41 +/- 0.81 among the cases. Positive EGFR expression correlated strongly with differentiation of the tumour, poorly differentiated tumours showing a higher positivity. EGFR positivity also showed good correlation with metastasis as well as with the invasiveness of the tumour. Ki 67 labelling indices correlated significantly with metastatic status, microscopic types and degree of differentiation of the tumour. A strong correlation was observed between AgNOR counts and metastasis as well as the microscopic type of the tumour. EGFR expression correlated strongly with Ki 67 scores and weakly with AgNOR counts among the patients of gastric carcinoma.     

Expression of bcl-2, p53 and Ki-67 in arsenical skin cancers

To investigate the regulation of apoptosis and proliferation in arsenic-induced skin cancers, we examined the expression of bcl-2, p53, and Ki-67 using immunohistochemical staining. Thirty patients with Bowen's disease (BD), ten with basal cell carcinoma (BCC), eight with squamous cell carcinoma (SCC) and eleven of perilesional normal skin (PLN) of the non-sun exposure sites from endemic area were examined. The results showed that: 1) bcl-2 was expressed in all of the BCC homogeneously, in none of the SCC, and in 12/30 of the BD focally or homogeneously; 2) p53 was expressed in all of the arsenical skin cancers with a labelling index of 75 +/- 14% of BD, 50 +/- 17% of BCC, 61 +/- 15% of SCC, and also in all of the perilesional normal skin with a labelling index of 55 +/- 24%; 3) Ki-67 was expressed in all of the skin cancers with labelling index of 58 +/- 17% of BD, 12 +/- 7% of BCC, 47 +/- 21% of SCC, and in 9/11 of PLN with a labelling index of 41 +/- 24%. Expression of bcl-2 in BCC or BD is related to the phenotype of germinative basal cell. The constant expression of bcl-2 i early dysplastic cells of BD and the earliest expression of P53 in the basal cells of perilesional normal skin indicate that the initial step of arsenic-induced carcinogenesis is from the basal germinative cells. There is no mutual relationship between bcl-2, p53 or Ki-67 expression in any type of the arsenical skin cancers, but there is a positive correlation between p53 and Ki-67 expression identified in perilesional normal skin. BD had the highest labelling index of p53 and Ki-67.     

DNA ploidy, P53 expression, and cellular proliferation in normal epithelium and squamous dysplasia of non-cancerous and cancerous human oesophagi

Ki67 expression, S-phase fraction, p53 immunoreactivity and DNA content were examined in morphologically normal mucosa and squamous dysplasia of both cancerous and non-cancerous human oesophagi in order to understand possible early events in the development of esophageal squamous cell carcinoma. 103 different foci from cancerous esophagi including 17 non-pathological epithelium, 10 mild, 17 moderate and 15 severe dysplasia, 14 intraepithelial carcinomas and 30 invasive squamous cell carcinomas were examined. Also studied were 57 biopsy specimens from cancer-free individuals, including 12 normal epithelia, 15 oesophagitis, and 16 mild, 11 moderate and 3 severe dysplasia. Areas of squamous dysplasia from both cancer-free and cancerous oesophagi were morphologically indistinguishable and both demonstrated increased cellular proliferation compared to normal or non-pathological epithelia. However, squamous dysplasia in cancerous oesophagi demonstrated significantly larger ki67 labelling indices and smaller S-phase fractions than dysplasia in cancer-free patients. Squamous dysplasia in cancerous and non-cancerous oesophagi demonstrated an non-diploid DNA histogram in 67.9% and 43.3% respectively. However, dysplasia from cancer-free individuals demonstrated a non-diploid pattern with one or more peaks (Type I non-diploid histogram) and that from oesophageal cancer patients predominantly exhibited non-diploid histograms without any distinctive peaks (Type II non-diploid histogram). Significant differences in the frequency of p53 positive foci were observed between dysplasia of cancer-free (23.3%) and cancerous (56.8%) oesophagi. IN cancerous oesophagi, dysplasia associated with Type II non-diploid histograms had a significantly larger number of p53-positive foci than those with diploid histograms or Type I non-diploid histograms. These results indicated that the biological features of squamous dysplasia were different between cancerous and non-cancerous human oesophagi despite indistinguishable morphological features. In addition, the combination of p53 immuno-histochemistry and DNA ploidy analysis may contribute to identify possible high-risk squamous dysplasia of the oesophagus.     

Cell proliferation of breast cancer evaluated by anti-BrdU and anti-Ki-67 antibodies: its prognostic value on short-term recurrences

The prognostic value of breast cancer proliferative activity was evaluated in 385 women operated for primary, non-metastasised mammary carcinoma. Cell kinetics was measured using two immunohistochemical techniques. Cells in S-phase of cell cycle were labelled in vitro by incubation of fresh tissue fragments with 5-bromo 2-deoxyuridine (BrdU), a thymidine analogue. Nuclei of cells in active DNA synthesis were stained by an anti-BrdU monoclonal antibody (Mab). Cells in interphase and mitosis were detected with Ki-67, a Mab that is known to react with a nuclear antigen present in G1/S/G2/M phases of cell cycle, but not in resting cells. This reagent provides a means of evaluating the growth fraction of neoplastic cells. BrdU was incorporated in a proportion of tumour cells ranging from 0.1 to 65.5% (median 6.8%). In the panel of tumours presented in this report the median percentage of Ki-67 positive cells (Ki-67 score) was 9.0% (range 0.1-77%). The relationship between disease-free survival (DFS), BrdU labelling index, Ki-67 score and 13 different clinico-pathological variables was investigated by multivariate analysis, using the Cox proportional hazards model. Axillary node status (P = 0.009) and Ki-67 score (P = 0.038) emerged as independent prognostic factors. Nodal status and tumour growth fraction allowed division of patients into groups at different risk of relapse: tumours with a proliferative index below the median value showed a lower recurrence rate than tumours with a high proliferative activity (P < 0.001). In particular, no relapse occurred in pN0 patients bearing carcinomas with a Ki-67 labelling < 9.0% 4 years after surgery. These findings suggest that the evaluation of proliferative activity in breast cancer enhances the probability of correctly predicting outcome after surgery and could be of assistance in the planning of adjuvant therapies.     

Sex-role reversal and the absence of extra-pair fertilization in Wilson''s phalaropes

Human endometrial leukocytes undergo regular cyclical changes during the menstrual cycle, with a striking increase in the phenotypically unusual population of CD56+ CD16- endometrial granulated lymphocytes (eGLs) in the late secretory phase and early pregnancy. The factors that regulate this increase in eGL numbers are unclear; their unusual morphology, however, has led to the suggestion that they undergo apoptosis at the end of the menstrual cycle. Apoptosis, bcl-2 expression, and proliferative activity were examined in the stroma of normal cycling, progesterone-treated, and early-pregnancy endometrium. The expression of bcl-2 and the Ki67 proliferation marker by highly purified (> 98% CD56+) eGLs from endometrium during the menstrual cycle and from first-trimester decidua was also studied. Apoptotic cells were rarely observed in the endometrial stroma of any of the samples examined. Stromal bcl-2 expression, however, increased from the proliferative to the premenstrual phase, and double immunohistochemical labeling demonstrated large numbers of bcl-2+ CD56+ eGLs. In contrast, Ki67 expression was high in the endometrial stroma during the proliferative phase, fell during the secretory phase, and rose again premenstrually, because of expression by eGLs. Isolated CD56+ eGLs also showed high bcl-2 and Ki67 expression at the end of the menstrual cycle. Unlike premenstrual endometrium, progesterone-treated endometrium and first-trimester decidua contained few proliferating cells, expressed high levels of bcl-2, and showed no evidence of apoptosis. Thus, eGLs do not undergo apoptosis in premenstrual endometrium, and their regulatory mechanisms remain to be clarified.     

Fibroblasts can modulate the phenotype of malignant epithelial cells in vitro

An organotypic, tridimensional cell culture, also called a raft system, was used to study the influence of fibroblasts on epithelial carcinogenesis in a cell line derived from laryngeal squamous cell carcinoma and harboring a mutated p53. Differences between the effects of normal fibroblasts and those of tumor-derived fibroblasts were compared by means of fibroblasts taken from the normal skin and from the tumor of a cancer patient and cultivated with epithelial carcinoma cells in an organotypic culture. To study cell contact-mediated changes, the fibroblasts were either simply embedded in collagen matrix or additionally brought into direct contact with epithelial cells. Control epithelial cells were cultivated without any fibroblasts in an organotypic model. A protein panel [p53, p21, PCNA, bcl-2, Ki67, total cytokeratin (CK), CK 8, CK 10, CK 17, CK 18, CK 19, vimentin] involved in cell cycling and epithelial differentiation was assessed immunocytochemically in all organotypic cultures with fibroblasts, in tumor cells cultivated as a monolayer, and in the original tumor sample. The most dysplastic phenotype was obtained when tumor-derived fibroblasts were used in direct contact with epithelial cells, whereas the most benign phenotype was seen when skin fibroblasts had no contact with them. The intensive staining seen for p53 can be explained by p53 mutations also reflecting the weak expression of p21 and abundant expression of PCNA. The intensive Ki67 staining seen in all sections paralleled that of PCNA and marked active cellular proliferation. The CK staining pattern seen in cultured epithelia toward embryonic CKs, CK 8 and CK 18, suggested a simple epithelial phenotype. CK 19 was found only in the epithelium where no direct contacts had occurred. Vimentin expression increased when the raft epithelium was shifting toward a more benign phenotype. The results stress the importance of the origin of fibroblasts as well as the role of direct cellular contacts in modifying the epithelial phenotype even when the epithelial cells are malignant.     

Transcatheter regional urokinase therapy in the management of inferior vena cava thrombosis

The prognosis of colorectal cancer has not significantly changed during the last 30 years. While evaluation of tumour cell proliferation may provide prognostic information, results obtained so far have been contradictory Heterogeneity in tumour cell proliferation may explain these contradictions. With in vivo injection of iododeoxyuridine (IdUrd), estimation of labelling index (LI), S-phase transit time (Ts) and potential doubling time (Tpot) may be performed from a single sample. A total of 109 colorectal cancers were studied after in vivo injection of IdUrd before surgical removal. From each cancer, four to eight samples were processed for both flow cytometrical (FCM) and immunohistochemical (IHC) visualization of IdUrd incorporation. LI/IHC was morphometrically quantified at both the luminal border and the invasive margin of these tumours. LI was significantly higher at the luminal border compared with the invasive margin, although they were correlated with each other. Using combined IHC and FCM methods, rapidly growing colorectal cancers (high LI and/or low Tpot) showed an increased survival (significant for LI at the invasive margin and for Tpot at both the invasive margin and the luminal border) in the entire unselected material and for radically removed Dukes' B tumours. FCM data alone did not discriminate for survival, with the exception of Ts in diploid and radically removed Dukes' B tumours.     

Expression of nucleolar protein p120 in human lung cancer: difference in histological types as a marker for proliferation

The function of proliferation-associated nucleolar protein p120 is unclear. A recent report that a yeast protein, NOP2, 67% homologous to human p120, is up-regulated during the onset of growth and influences the morphology of the nucleolus supports the notion that this protein could serve as a marker for proliferation in neoplastic cells. Lung cancer is characteristic in that different histological types show different biological features. We attempted to evaluate the levels of p120 expression in resected human lung cancer tissues of different histological types and the relation of p120 expression and cell proliferation using human lung cancer cell lines. When 37 frozen specimens of human lung cancer and normal lung were stained with a p120 monoclonal antibody, the nucleoli of cancer cells were positively stained, whereas a few macrophages in normal lung revealed only weak staining. The labeling index of p120 in squamous cell carcinoma (67.7 +/- 12.4%) was significantly higher than that in adenocarcinoma (35.3 +/- 12.6%) or in large cell carcinoma (30.1 +/- 17.3%; P < 0.01). In six human lung cancer cell lines and one normal lung fibroblast cell line cultured in vitro, there was a significant correlation between S-phase fraction and p120 mRNA (r = 0.851, P < 0.02)/p120 protein (r = 0.869, P < 0.01) or between doubling time and p120 protein (r = -0.928, P < 0.01). In the context of the reports that indicate higher [3H]thymidine incorporation and shorter doubling time in the squamous cell carcinoma, these results indicate that p120 can be a marker for proliferation in human lung cancer cells in vivo and in vitro, and that it has an important function in the cell cycle of tumor proliferation.     

Epithelial proliferation and hormone receptor status in the normal post-menopausal breast and the effects of hormone replacement therapy

The proliferation rate (as assessed by Ki67 expression) and expression of oestrogen-regulated progesterone receptor (PR) was studied in normal post-menopausal breast epithelium. Normal breast epithelium from patients receiving hormone replacement therapy (HRT) at the time of surgery containing either oestrogen alone (E2) or oestrogen and progesterone combined activities (E2 + P) was also studied, as HRT has been linked to an increased breast cancer risk. Samples of breast tissue, containing normal epithelium, from 185 patients undergoing surgery for benign or malignant disease were immunocytochemically stained for PR and Ki67. The percentage of labelled cells was expressed as the labelling index (LI). The median Ki67 LI in normal post-menopausal breast epithelium was 0.19 and median PR LI was 4.75, and both were unaffected by patient age, duration of menopause or if the tissue sample originated from a breast with benign or malignant disease. Proliferation did not alter significantly in patients taking HRT (P = 0.61); however, PR expression was up-regulated in both E2 and E2 + P users (P = 0.01). The dose and duration of HRT had no effect on either parameter. A possible attenuation of sensitivity to oestradiol-induced proliferation but not to PR expression occurs in the post-menopausal breast.     

Variation of H1(0) content throughout the cell cycle in regenerating rat liver

Histone H1(0), a differentiation-specific member of the histone H1 family, accumulates in cells during the terminal phase of cell differentiation, in tissues composed of arrested cells or cells exhibiting little proliferation. Moreover, the induction of cell proliferation in vivo, i.e., after partial hepatectomy, is accompanied by a decrease in H1(0) content. These observations suggest that H1(0) may be involved in the arrest of cell proliferation in vivo. In order to investigate this possibility, we took advantage of the fact that after partial hepatectomy the initiation of cell division is not synchronous. The strategy was to know, at the level of a single cell, whether H1(0) decreases prior to the initiation of the S phase or whether a cell can initiate DNA replication having a significant amount of H1(0) in the nucleus. We defined new protocols to analyze H1(0) content and cell proliferation at the level of a single cell, both in situ and by flow cytometry. The simultaneous determination of the relative amount of H1(0) and the position of cells in the cell cycle showed that no significant difference in H1(0) content was detected in cells actively replicating their DNA compared to nondividing cells. These observations have been confirmed by the successive immunodetections of H1(0) and BrdU in situ on the same cells. Therefore, we show here that in vivo, cells can initiate DNA replication with significant amounts of H1(0) and that the decrease of H1(0) is not a prerequisite of cell division. We propose that the accumulation of H1(0) is not related to the arrest of cell proliferation, but is controlled in such a manner that the protein accumulates in slowly dividing cells and decreases in rapidly growing cells.     

On the release of glutamate and aspartate in the basal ganglia of the rat: interactions with monoamines and neuropeptides

Experiments were designed to determine the in vivo cell-cycle phase of lymphocytes in secondary lymphoid follicles, and whether B-cell proliferation and apoptosis occur within follicular dendritic cell (FDC)-associated clusters. Using frozen serial sections of human tonsils, lymphoid follicles were stained to reveal histone H3 mRNA, as an S-phase marker, using in situ hybridization, and stained immunohistochemically with antibodies against cyclin E as a late G1 phase marker, cyclin B1 and p34cdc2 as S-G2-M phase markers, and Ki-67 as a marker of cycling cells. Each LF was divided into five zones: mantle zone, outer zone, apical light zone, basal light zone and dark zone, with the help of haematoxylin and eosin staining, and a CD23 immunostain. The rate of occurrence of positively labelled cells was calculated by dividing the number of positive cells by the number of all cells in each zone. The cells that were positive for cyclin E, histone H3 mRNA, cyclin B1, p34cdc2, and Ki-67 were found most frequently in the dark zone (54.5 +/- 6.6%, 22.0 +/- 5.7%, 36.7 +/- 14.5%, 40.0 +/- 10.2%, and 59.0 +/- 13.4%, respectively), followed by the outer zone (52.7 +/- 7.8%, 14.9 +/- 4.1%, 22.9 +/- 9.7%, 24.9 +/- 7.9%, and 44.6 +/- 12.3%, respectively), showing that both the outer zone and the dark zone contain many proliferating lymphocytes. Furthermore, FDC-associated clusters and free lymphocytes were obtained from enucleated germinal centres, using enzymatic digestion. The rates of occurrence of cells that were positive for cyclin B1 and Ki-67 within the clusters (7.2 +/- 1.9% and 37.9 +/- 10.5% respectively) were significantly lower than those of free lymphocytes outside the clusters (22.2 +/- 4.0% and 62.8 +/- 14.0%, respectively). The rates of occurrence of apoptotic bodies and cells within the clusters, as detected by in situ tailing or in situ nick translation (0.2 +/- 0.4% and 0.4 +/- 0.4%, respectively) were significantly lower than those outside the clusters (1.1 +/- 0.3, 1.6 +/- 0.5%, respectively). These results suggest that FDC-associated clusters are not the site of proliferation, and that they rarely contain apoptotic bodies and cells of B lymphocytes.     

Cerebral cortex regional blood flow and tissue oxygen tension during the trigeminal depressor response in rabbits

The paradox of pancytopenia despite cellular bone marrows (BM) was investigated in 120 patients with myelodysplastic syndromes (MDS). Detailed cell cycle kinetics were examined following in vivo infusions of iodo--and/or bromodeoxyuridine (IUdR/BrdU), while the incidence of apoptosis was measured by in situ end labeling (ISEL) of fragmented DNA. Results showed that MDS are highly proliferative disorders with an equally high incidence of apoptotic intramedullary cell death accounting for the paradox of cellularity/cytopenia. By double-labeling BM biopsy sections for ISEL/BrdU we found the peculiar situation of "signal antonymy" where S-phase cells were frequently apoptotic, a phenomenon so far only seen in MDS biopsies. The cause-effect relationship of this excessive proliferation/apoptosis is discussed at length.