bluuebunny/crossref_metadata_2025_split huggingface dataset:
Variation in expression of genes used for normalization of Northern blots after induction of cell proliferation
Authors: Goldsworthy S. M. , Goldsworthy T. L. , Sprankle C. S. , Butterworth B. E. 
Published: May 2008

AbstractQuantitative knowledge of gene expression can provide valuable information for understanding the action of chemicals that alter cell proliferation and cancer. Accurate quantification of mRNA levels requires the normalization of the gene of interest to a gene with transcriptional levels that do not vary through the cell cycle or with a particular treatment. Changes in expression were examined in proliferating or non‐proliferating rat liver for three constitutively expressed ‘housekeeping’ genes commonly used to normalize mRNA levels from Northern blots. In addition, a direct method of quantifying poly(A)+ mRNA by hybridization with a radiolabelled polythymidylate—poly(T)—probe was compared with traditional methods. Hepatocyte cytolethality and a subsequent wave of hepatocyte proliferation were induced in male Fischer‐344 rats by treatment with a single gavage dose of carbon tetrachloride. Induced cell proliferation peaked at 48 h after treatment. Expression of the housekeeping genes actin, glyceraldehyde‐3‐phosphate‐dehydrogenase (GAPDH) and albumin, as well as the proto‐oncogene H‐ras, was determined by Northern blot analysis at times from 0.5 h to 4 days after treatment. Time‐dependent changes were observed in the expression of these genes relative to the levels observed in the untreated control animals. Actin expression peaked at 3.4‐fold over control and GAPDH expression was increased by 1.9‐fold over control. Albumin mRNA levels varied the least, 1.4‐fold over control, indicating that this gene is more appropriate than actin or GAPDH for normalization of proto‐oncogene expression under experimental conditions that induce cell proliferation in rat liver. The direct quantification of poly(A)+ mRNA using a poly(T) probe was not influenced by the induction of cell proliferation. This method may be useful when the expression of housekeeping genes is affected by treatment.
Mechanism of differentiation of human erythroleukaemic cell line K562 by hemin
Authors: Baliga B. S. , Mankad M. , Shah A. K. , Mankad V. N. 
Published: May 2008

AbstractThe human erythroleukaemic cell line K562, in response to various chemical agents, undergoes differentiation and exhibits exclusive production of fetal and embryonic haemoglobins. In this study we have compared the efficiency of natural growth factors interleukin‐3 and erythropoietin and three chemical inducers such as dimethyl sulfoxide (DMSO, 1.9%), phorbol‐12‐myristate‐13‐acetate (PMA, 50 ng/ml) and hemin (25 μm) on growth and differentiation of these cells. Erythropoietin significantly stimulated the growth of K562 cells (P<0.0001), while interleukin‐3 did not (P= 0.2783). However, neither of these growth factors individually or together induced differentiation of K562 cells. Hemin appears to be more efficient than DMSO or PMA in differentiation of K562 cells as measured by benzidine positive cells (70% or more). The differentiation of K562 cells by hemin occurs independently of protein kinase‐C activation and the arrest of DNA synthesis. In contrast, hemin significantly stimulated RNA and protein synthesis (P<0.0001) as measured by [3H]‐uridine and [3H]‐leucine incorporation respectively. Analysis of hemin‐treated K562 nuclear extract on sodium dodecylsulphate gel electrophoresis showed that one protein band of molecular weight 70 kDa decreased after 48 h of incubation in the presence of 25 μm hemin. The disappearance of this protein can be prevented by cycloheximide (100 μg/ml) and actinomycin D (0.1 μg/ml) and thus indicating that the removal of 70 kDa protein seems to be dependent on RNA and protein synthesis. The regulatory role of 70 kDa protein in hemin‐induced differentiation of K562 cells is discussed.
Changes in the extents of viable and necrotic tissue, interstitial fluid pressure, and proliferation kinetics in clone A human colon tumour xenografts as a function of tumour size
Authors: Leith J. T. , Michelson S. 
Published: May 2008

Abstract. Total, viable and necrotic tumour tissue, tumour cell yields, and colony forming efficiencies were measured in clone A human colon tumour xenografts as neoplasms grew from about 100 mm3 to about 6000 mm3. The volumes of the total, viable and necrotic compartments were fit using the Verhulst equation to obtain estimates of growth rates and maximal sizes of the various compartments (carrying capacities). Additionally, at four discrete tumour volumes (250, 850, 2500 and 5500 mm3), hypoxic percentages, proportions of parenchymal tumour and host cells, interstitial fluid pressures, and proliferation kinetics including measurements of apoptosis were determined.There were interesting relationships between the shapes of the curves for total, viable and necrotic tissue to some of the other endpoints measured. Specifically, the volumetric growth curves for the total and viable tumour tissue compartments were identical to a volume of approximately 1000 mm3, but diverged at larger sizes, with the viable cell compartment exhibiting a smaller carrying capacity. The shape of the growth curve for the necrotic compartment exactly mimicked that for the total volume compartment, but was delayed in time by about 21 days. Similarity in shape to that of the overall tumour volume/necrotic volume curves was also seen for the curve for the increase in interstitial fluid pressure, and for the increase in the size of the host cell compartment. In contrast, the growth of the hypoxic compartment and of the parenchymal tumour cell compartment were similar in shape to that of the viable compartment. These data indicate that these compartments are functionally linked.Marked changes in cell kinetic parameters occurred as tumour size increased from 250–5500 mm3. The labelling index and growth fractions decreased from 0.256–0.125, and 0.77–0.40 respectively, and the cell loss factor increased from 0.52–0.74. The volumetric and potential doubling times increased from 4.3–17.6 and 2.1–4.6 days respectively. The cell kinetic changes could not be clearly related to the changes in shape of either the overall tumour volume or the viable tumour volume.
FB1, a monoclonal antibody reacting with a keratin 14 epitope, stains only a small subset of psoriatic basal keratinocytes
Authors: Parent D. , Golstein J. , Mairesse N. , Bernerd F. , Faverlyt D. , Robben H. , Ramaekers F. , Heenen M. 
Published: May 2008

AbstractA murine monoclonal antibody, FB1, reacted with the basal keratinocytes of human stratified epithelia. One‐dimensional and two‐dimensional immunoblotting assays, performed on keratins extracted from HaCat cells and normal human keratinocytes, showed that FBI recognizes K14. When LL002, another K14 monoclonal antibody is added, the FB1 stained area in the 2D‐immunoblot seems to cover a fraction of the LL002 spot. Immunohistochemical data obtained from studies on normal human tissues supported the K14 specificity of FB1, but when compared with two other monoclonal antibodies, LL002 and RCK107 reacting with K14, some differences appeared. These differences were mainly seen in sweat glands, hair follicles, psoriatic epidermis and salivary glands. In psoriatic epidermis, FB1 showed a heterogeneous pattern of staining of the basal cell compartment. Intense reactivity was only observed at the bottom of the rete ridges. Staining diminished and finally disappeared in the basal cells above the dermal papillae. This observation supports the view that an increased germinative cell population in psoriasis involves a partially differentiated amplifying compartment in which the number of cell divisions is increased.
Gompertzian re‐evaluation of the growth patterns of transplantable mammary tumours in sialoadenectomized mice
Authors: Bassukas I. D. 
Published: May 2008

AbstractThe method of the recursion formula of the Gompertz function (Bassukas & Maurer‐Schultze 1988) has been applied to analyse tumour growth data taken from the literature; namely the growth perturbation of transplantable mammary tumours in sialoadenectomized mice with or without subsequent epidermal growth factor substitution (results on two mouse strains, C3H or SHN, have been reported; Inui, Tsubura & Morii 1989). The recursion formula of the Gompertz function fits growth curves to all seven sets of data well (P > 0.05 for lack of fit test). The growth pattern of the tumours in the unperturbed hosts is Gompertzian and does not change if tumours are transplanted in sialoadenectomized mice, although the starting specific growth rate decreases in C3H mice. However, if sialoadenectomy is carried out after tumour inoculation, a complex alteration of the tumour growth evolves: tumour growth does not simply decelerate but it also shifts from the conventional Gompertzian to an exponential or even ‘hyperexpo‐nential’ growth pattern, i.e. with an accelerating specific growth rate. Some theoretical mechanisms of this alteration, as well as the differences between the present Gompertzian analysis and a previously published Verhulstian analysis of part of the same data (Leith, Harrigan & Michelson 1991), are discussed. It is concluded that the quantitative analysis of tumour growth patterns by the method of the difference equation of the Gompertz function presently applied may substantially contribute to the improvement of the interpretation of perturbations of tumour growth‐irrespective of their genesis. In contrast to the application of some a priori fixed growth function, e.g. the Verhulstian one, the present method can quantitatively interprete different growth patterns and their classification on the basis of linear regression analysis.
The effects of vinblastine in assessment of the influence of age on proliferative activity of murine palate and footpad epithelium
Authors: Berg J. H. , Hill M. W. 
Published: May 2008

AbstractData concerning changes in the rate of cell proliferation of stratified epithelia with increasing age are conflicting. In the present study young (3‐month‐old) and old (22‐month‐old) C57B1/6NNia male mice were injected intraperitoneally with 2, 3, 4 or 8 mg vinblastine sulfate/kg body weight and killed after 1.5, 3, 4.5 or 6 h. The number of arrested metaphase figures per 1000 basal cells was counted in histological sections. Data were analysed using a multivariate analysis of variance. There was a significant difference between the accumulation of mitotic figures in footpad epidermis and palate epithelium and both tissues contained an increased number of mitotic figures with increasing periods of accumulation at all dose levels. In the footpad epidermis neither the age of the animal nor the dose of vinblastine had a significant effect on the number of mitotic figures. In contrast, for palate epithelium the accumulation of mitotic figures was significantly less in the old mice compared with the young mice and at a dose of vinblastine of 2mg/kg compared with the higher doses. There was a statistically signifycant interaction between the dose of vinblastine and its period of action. It was concluded that the different tissues manifest a differential sensitivity to vinblastine and that only palate epithelium showed a significant reduction in proliferative activity with age.
Comparison of monoclonal antibodies PC 10 and MIB 1 on microwave‐processed paraffin sections
Authors: Kubbutat M. H. G. , Cattoretti G. , Gerdes J. , Key G. 
Published: May 2008

Abstract. Antibodies against the proliferation‐associated nuclear antigen (PCNA) and against the Ki‐67 protein are widely used as operational proliferation markers in human tumour diagnostics. The original Ki‐67 antibody had the inherent drawback in that it could only be used when fresh‐frozen material was available. The antibody PClO was supposed to offer the advantage that it could be applied on formalin‐fixed and paraffin‐embedded tissues. However, in cases in which the formalin fixation exceeded 4 h, PC10 staining proved to be inconsistent and often failed.The aim of this study was to compare a recently prepared Ki‐67 equivalent monoclonal antibody (MIB 1) and PC10 in routinely fixed histopathological material using antigen retrieval by microwave processing.Antibody MIB 1 stained the nuclei of cells known to belong to the proliferative compartments in microwave‐processing paraffin sections of formalin‐fixed tissues. Quiescent cells were consistently negative for MIB 1 staining. In contrast, PC10 was positive in almost all nuclei of different tissues in microwave‐treated paraffin sections. Thus, antigen retrieval by microwave processing is beneficial for the detection of the Ki‐67 protein in paraffin sections, whereas it is not needed for the detection of the PCNA.
Cell cycle of neural crest cells in the early migratory stage in vivo
Authors: Paglini M. G. , Rovasio R. A. 
Published: May 2008

Abstract. The fact that directional migration of neural crest cells (NCC) in vivo occurs in narrow pathways at high cell density, together with our preliminary results showing their high proliferative behaviour, supports the view that a ‘population pressure’ could be an important factor in the mechanism of early dispersion of NCC.The purpose of this work was to establish the cranial proliferative pattern of chick embryo NCC during their early migratory stage in vivo. Growth rates and parameters of cell cycle were obtained from cell populations at several cephalic levels by means of autoradiography after labelling with [3H]dT. The labelled cell index of NCC (Forebrain, 0.288; Midbrain, 0.206; and Hindbrain, 0.134) was significantly greater than in other cells populations (e.g. for the neural tube cells: 0.085, 0.030, and 0.031, respectively). The cell generation time was the shortest in NCC (16 h), compared to ectoderm (33 h), mesoderm (58 h) and endoderm (72 h). The duration of the cell cycle phases for NCC were: M, 0.29 h; G1, 11.23 h; S, 3.40 h; and G2, 1.02 h.These quantitative results show that NCC have the greatest proliferative rate in young chick embryos. In relation to cranial regions, the data are consistent with the idea that, in the early migratory phase of this cell population in vivo, migration is in part driven by population pressure.
Relationship between epidermal growth factor receptor levels, autophosphorylation and mitogenic‐responsiveness in normal mouse mammary epithelial cells in vitro
Authors: McIntyre B. S. , Birkenfeld H. P. , Sylvester P. W. 
Published: May 2008

AbstractMammary epithelial cells were isolated from mid‐pregnant BALB/c mice, grown within collagen gels and maintained on DME/F12 (1:1) media containing 10% bovine calf serum and 10 μ/ml insulin. Initial time‐course and dose‐response studies showed that epidermal growth factor (EGF)‐induced autophosphorylation of the EGF‐receptor (EGF‐R) in these cells was maximal 5 min after exposure to 75 ng/ml EGF. Mammary epithelial cells displaying little or no growth during their first 2 days in primary culture cells were found to contain low levels of EGF‐R. However, EGF‐induced autophosphorylation of the EGF‐R in these cells was extremely intense. Subsequent studies demonstrated that during the proliferative and plateau phases of growth, EGF‐R levels progressively increased, while conversely EGF‐induced autophosphorylation of the EGF‐R decreased over time in primary culture. These results demonstrate that EGF‐R levels and autophosphorylation do not show a direct correlation with mammary epithelial cell mitogen‐responsiveness. Intense EGF‐R autophosphorylation appears to be required for initiating growth, but sustained mammary epithelial cell proliferation occurs when EGF‐R autophosphorylation is low. This inverse relationship between EGF‐R levels and autophosphorylation may reflect changes in receptor affinity and function during the various phases of mammary epithelial cell growth in primary culture.
Labelling DNA strand breaks with BrdUTP. Detection of apoptosis and cell proliferation
Authors: Li X. , Darzynkiewicz Z. 
Published: May 2008

Abstract In situ presence of numerous DNA strand breaks is a typical feature of apoptotic cells. Selective DNA strand break induction by photolysis (SBIP) at sites that contain incorporated halogenated DNA precursors has recently been proposed as a method of analysing DNA replication. Detection of DNA strand breaks, thus, enables one to identify apoptotic and/or DNA replicating cells. The current methods for DNA strand break labelling rely on the use of exogenous terminal deoxynucleotidyl transferase which either directly attaches the fluorochrome conjugated triphosphodeoxynucleotides to 3′OH ends in the breaks, or indirectly labels 3′OH ends with digoxygenin or biotin conjugated triphosphodeoxynucleotides. A limitation of these methodologies, especially restricting their routine application in the clinic, is high cost of reagents. In the present study we have tested whether relatively simple compound BrdUTP, which is approximately three orders of magnitude less expensive than dUTP conjugated to digoxygenin, can be used as marker of DNA strand breaks. Apoptosis of HL‐60 cells was induced by DNA topoisomerase I inhibitor camptothecin. The incorporated BrdUTP was detected by fluoresceinated anti‐BrdUrd MoAb. Cellular fluorescence was measured by flow cytometry as well as by Laser Scanning Cytometer (LSC). The data show that intensity of DNA strand break labelling with BrdUTP was nearly four‐ and two‐fold higher than that obtained with the indirect labelling using biotin‐ or digoxygenin‐conjugated dUTP, respectively, and over eight‐fold higher than in the case of direct labelling with the fluorochrome (fluorescein or BODIPY)‐conjugated deoxynucleotides. The increased labelling of DNA strand breaks with BrdUTP may reflect more efficient incorporation of this precursor by terminal transferase, compared to the nucleotides with bulky fluorochrome conjugates. DNA strand break labelling with BrdUTP, thus, offers a possibility of more sensitive (and at lower cost) detection of apoptotic or DNA replicating cells, compared to the alternative methods of DNA strand break labelling.
Cell proliferation in the subependymal layer of the adult mouse in vivo and in vitro
Authors: Hauke C. , Ackermann I. , Korr H. 
Published: May 2008

AbstractPulse labelling experiments with [3H] thymidine (dT) and double labelling experiments with [3H]dT and bromodeoxyuridine (BrdUrd) were carried out on cells of the subependymal layer in the brain of adult normal mice in vivo, in vivo/in vitro and in vitro. The results should (i) lead to information about cell cycle parameters of these cells in the brain of adult mice, since these cells have been studied mostly in the rat brain up to now and (ii) answer the question whether results concerning cell proliferation obtained in vivo correspond with those from brain slices incubated in vitro with or without prelabelling in vivo. In vivo an LI of 20.2 ± 2.7% (x̄± SEM) and Ts= 7.2 ± 0.7h were found. Furthermore, grain count halving experiments led to a surprisingly short cycle time (Tc) of 11.2–14.2 h. The longer Tc values (18–20 h) reported in the literature for subependymal cells in the rat brain seem to be due to evaluations of different areas around the lateral ventricle without considering the migrating behaviour of these cells which is quite different regionally. The in vitro studies (with or without prelabelling in vivo) showed a significantly reduced LI due to the fact that about 20% of the S phase cells, possibly lying in the middle of S, stopped further DNA synthesis after transfer to culture. This was shown by comparing the cell fluxes at the G1/S and S/G2 borders of in vivo vs. in vitro studies.
In vitro bromodeoxyuridine‐labelling of single cell suspensions: effects of time and temperature of sample storage
Authors: Carbajo‐Pérez E. , Carbajo S. , Ciudad J. , López A. , Dolbeare F. , Orfao A. 
Published: May 2008

AbstractThe present study was aimed to explore how the in vitro BrdUrd‐labelling of rat thymocytes might be affected by both the time elapsed between obtaining the sample and the beginning of the labelling (0, 15, 30 or 60 min) and the effect of the temperature of storage (4°C versus room temperature). Single cell suspensions obtained after in vivo labelling with BrdUrd were used as controls. The S phase fraction was calculated by flow cytometry both according to BrdUrd‐immunolabelling and DNA content. Immediate incubation with BrdUrd after the sample was obtained resulted in a slight decrease of the proportion of S phase cells analysed either according to DNA content or to BrdUrd‐immunolabelling. Regardless of storage‐temperature, the S phase fraction decreased in samples kept for 15 min or more before BrdUrd incubation. No BrdUrd‐positive cells were detected in samples stored for 60 min at room temperature. This effect was related to temperature since positive cells were found when the samples were kept at 4°C during the same time period. Our results suggest that during in vitro incubation a relative loss of S phase cells exists and that a delay beyond 15 min between obtaining the sample and the in vitro labelling seriusly compromises the results of this technique.
Comparison of proliferating cell nuclear antigen (PCNA) staining and BrdUrd‐labelling index under different proliferative conditions in vitro by flow cytometry
Authors: Lohr F. , Wenz F. , Haas S. , Flentje M. 
Published: May 2008

AbstractPC10 is a monoclonal antibody against proliferating cell nuclear antigen (PCNA). The staining pattern in immunochemistry depends on fixation and detergent extraction treatment. The aim of this study was to validate the flow cytometric PCNA assay against Bromodeoxyuridine‐labelling index (BrdUrd‐LI) under different proliferative conditions in vitro. Expression of PCNA in methanol fixed cells with, and without, prior detergent extraction with EDTA/Triton was compared to BrdUrd‐labelling index in NIH‐3T3 fibroblasts and human Caski tumour cells in exponential phase and under confluent conditions. Serum stimulation and serum starvation conditions were studied. The results for BrdUrd‐LI and PCNA‐index after extraction showed good correlation for 3T3 fibroblasts and for Caski cells, with some differences for serum withdrawn Caski cells. There was no correlation between the number of cells that were positive for PCNA without extraction and BrdUrd‐LI. Spheroid cells with G1‐DNA‐content showed an almost synchronous recruitment and progression through the cell cycle after trypsination and replating. Tightly bound PCNA paralleled this synchronicity whereas total PCNA did not change significantly. The results demonstrate that immunochemical detection of non‐extractable PCNA‐index gives similar results as compared with BrdUrd‐labelling index under different proliferative conditions in vitro for different monolayer cell lines, whereas without extraction PCNA does not correlate with BrdUrd‐LI in these fast growing cell lines due to its long half‐life. PCNA expression parallels the progression through the cell cycle in V79 spheroids, a primitive model of tumour growth.