bluuebunny/crossref_metadata_2025 huggingface dataset:
Proliferating cell nuclear antigen and Ki‐67 antigen expression in human haematopoietic cells during growth stimulation and differentiation
Authors: Landberg* G. , Roos G.
Published: May 2008

Abstract. By flow cytometric dual parameter analysis of proliferating cell nuclear antigen (PCNA) and the Ki‐67 antigen a detailed cell cycle analysis can be performed. In this study the co‐ordinated expression of these two growth‐related antigens was investigated in human haematopoietic cells at entrance into the cell cycle as well as at exit from the cycle. In mitogen‐stimulated peripheral blood lymphocytes entering the first cell cycle, the Ki‐67 antigen was found to be expressed in S phase cells and not in G1 cells. Thus, the Ki‐67 antigen expression in PCNA‐positive S phase cells differed between continuously cycling cells and cells entering the cell cycle. Based on this difference, it was possible to visualize and evaluate the recruitment of cells into the first cell cycle from a resting stage. This new cell cycle parameter can give additional information concerning tumour growth. The Ki‐67 antigen was also studied during different stages of G1 and was found to be expressed at high levels in early G1 cells compared with other parts of G1.
Intratumoral heterogeneity of S phase transition in solid tumours determined by bromodeoxyuridine labelling and flow cytometry
Authors: Baisch H. , Otto U.
Published: May 2008

Abstract. Cell kinetics of human renal cell carcinomas xenotransplanted into nu/nu mice were analysed using the bromodeoxyuridine (BrdUrd) labelling method. Tumours were removed 0.5–14 h after injection of the BrdUrd solution. The tumour cells were stained with fluorescein isothiocyanate conjugated anti‐BrdUrd antibodies and propidium iodide (DNA content). From the flow cytometry data the relative movement was calculated. Relative movement data of variable intervals after BrdUrd labelling were subjected to a fit procedure using log‐normal distributions for S phase transition (Ts). The log‐normal distributions were modified by inflation factors in order to get extremely asymmetric distributions. The best fits to the experimental data were obtained using wide asymmetric Ts distributions, indicating that progression through S phase in solid human tumours is considerably heterogeneous. This implies that the potential doubling time (Tpot) is longer than calculated from a single measured relative movement value obtained a few hours after BrdUrd labelling.
Flow cytometric BrdUrd‐pulse‐chase study of heat‐induced cell‐cycle progression delays
Authors: Higashikubo R. , White R. A. , Roti J. L.
Published: May 2008

The flow cytometric, bromodeoxyuridine (BrdUrd)‐pulse‐chase method was extended by analysing five kinetic parameters to study perturbed cell progression through the cell cycle. The method was used to analyse the cell‐cycle perturbations induced by heat shock. Exponentially growing, asynchronous Chinese hamster ovary (CHO) cells were pulse labelled with BrdUrd and simultaneously heated at 43°C for 5,10 or 15 min. The cells were then incubated in a BrdUrd‐free medium and, at various times thereafter, were prepared for flow cytometry. Five compartments (BrdUrd‐labelled divided and undivided, and unlabelled G1, G1S, and G2) were defined in the resulting dual‐parameter histograms. The fraction of cells and the mean DNA content, when appropriate, were calculated for each compartment. The rates of cell‐cycle progression were assessed as time‐dependent changes in the fraction of cells in a given compartment and/or the relative DNA content of cells within a given compartment. Linear regression analysis of the data revealed two distinct modes of alteration in cell progression: 1 a delay in cell transit (either out of or into a given compartment), and 2 a decrease in the rate of cell transit. Hyperthermia produced a delay in the exit of cells from the G1 compartment of ≈ 16 min per minute of heat at 43°C with no threshold. In contrast, the delay in the exit of cells from all other compartments showed a threshold of from 3 to 5 min at 43°C. Above this threshold the delay in exit of cells from the BrdUrd‐labelled, undivided compartment was 25 min per minute of heat at 43°C. The more complex dose‐response function of this latter compartment may reflect the fact that it includes two cell‐cycle phases, S and G2+ M. The decrease in the rate of transit out of G2 for cells heated in G2 was significantly larger than that for any other compartment, consistent with previous studies, which showed a G2 accumulation following hyperthermia. These results indicate that heat exposure induces very complex alterations in cell‐cycle progression and that this flow cytometric method offers a straightforward approach for observing such alterations.
Evidence for altered cell‐cycle traverse of the non‐modal cells of the heteroploid MCa‐11 line
Authors: Dooley W. C. , Allison D. C. , Lin P. , Paul M.
Published: May 2008

Classic stem cell theory states that the growth of heteroploid cell populations is due to the proliferation of ‘main stemline’cells with modal DNA content and chromosome number. Cells with non‐modal DNA content and chromosome number are thought to be blocked and/or destroyed at mitosis. To test this, we studied two chromo‐somally stable cell populations (mouse bone marrow and WCHE‐5 cells) and one heteroploid, chromosomally diverse cell line (MCa‐11). The heteroploid MCa‐11 cells showed significant [3H]dT labelling for cells with DNA contents below the modal Go/G1 peak and above the modal G2 peaks (P<0.001). This was consistent with the presence of cells with the non‐modal DNA content that were engaged in replicative DNA synthesis. A percentage labelled mitosis analysis showed that MCa‐11 cells with non‐modal DNA content and chromosome number were able to complete mitosis, although with prolonged pre‐karyokinetic time. These results suggest that many non‐modal cells present in heteroploid cell populations are capable of continued proliferation.
Proliferation‐differentiation schema of murine haemopoietic lineages: a transient response
Authors: Novak J. P.
Published: May 2008

Abstract. A steady state model of haemopoiesis was derived recently from correlations of lineages (Novak JP & Necas E (1994). Proliferation differentiation pathways of murine haemopoiesis correlation of lineage fluxes. Cell Proliferation, 27, 597). Since the determination of some branching points was based on incomplete knowledge, the proposed schema was not necessarily a unique interpretation of the experimental data. This communication presents a first verification of the model usiing a comparison of calculated characteristics and observed data of post‐radiation recovery (following Necas E & Znojil V (1988) Bone marrow response to single small doses of irradiation: Implications for stem cell functional organization. Exp. Hematol. 16, 871). The measured population and S‐phase fraction of colony‐forming units‐spleen provided the entry variables and the time response of the granulocyte‐macrophage colony‐forming cells and neutrophils was calculated from the parameters of the model and some known kinetic coefficients. Good agreement with experiment is a strong indication that the earlier derived schema of haemopoietic pathways is truly representative of reality.
Monocytic differentiation of HL60 cells induced by potent analogs of vitamin D3 precedes the G1/G0 phase cell cycle block
Authors: Zhang F. , Godyn J. J. , Uskokovic M. , Binderup L. , Studzinski G. P.
Published: May 2008

Abstract. Differentiation of mammalian cells is accompanied by reduced rates of proliferation and an exit from the cell cycle. Human leukemic cells HL60 present a widely used model of neoplastic cell differentiation, and acquire the monocytic phenotype when exposed to analogs of vitamin D3 (VD3). The maturation process is accompanied by two blocks in the cell cycle: an arrest in the G1/G0 phase, and a recently described G2+ M block. In this study we have analyzed the traverse of the cell cycle phases of the well‐differentiating HL60‐G cells exposed to one of ten analogs of VD3, and compared the cell cycle effects of each compound with its potency as a differentiation‐inducing agent. We found that in general there was a good correlation between the effects of these compounds on the cell cycle and on differentiation, but the best cell cycle predictor of differentiation potency was the extent of accumulation of the cells in the G2 compartment. All analogs induced a marked decrease in the mitotic index, and polynucleation of HL60 cells was produced, especially by compounds which were effective as inducers of differentiation. Time course studies showed that induction of differentiation was accompanied by a transient increase of the proportion of cells in the G2+ M compartment, but preceded the G1 to S, and the G2 compartment blocks. These studies indicate that complex changes in the cell cycle traverse accompany, but do not precede, the acquisition of the monocytic phenotype by HL60 cells.
Renal transforming growth factor beta 1 production in neonate rats
Authors: Coimbra T. M. , da Silva C. G. A. , Lachat J. J.
Published: May 2008

Abstract. The newborn rat kidney is not fully developed until approximately 12 days after birth. In order to evaluate the possible role of Transforming Growth Factor beta 1 (TGF beta 1) in renal development we analyzed the mRNA level for TGF beta 1 in sixty Wistar rats aged 1,15 and 30 days. We also performed immunohislochemical studies to visualize the distribution of this peptide in the kidney of these rats using a TGF beta 1 antibody. The results show that the mRNA levels for TGF beta 1 are higher in the kidneys of the 1‐day‐old rats than in the 15 (1.4 fold) and 30‐day‐old rats (1.7 fold). The immunohistochemical reaction revealed the presence of TGF beta in the kidneys of the rats. The staining intensity was higher in the renal cortex than in the medulla. The data suggests that TGF beta may be important during kidney development.
Dose‐response of two cellular proliferation phenotypes produced by simian virus 40 large T antigen
Authors: Sladek T. L. , Fisher S. D. , Rubenstein B. N.
Published: May 2008

Abstract. A set of cell lines was constructed by infection of established murine fibroblasts with recombinant retroviruses encoding the simian virus 40 large T antigen (Tag) gene. By immunofluorescence flow cytometry, it was shown that these cell lines expressed Tag over at least a 20‐fold concentration range. Using these cells, the dose‐response relationship between Tag concentration and a phenotype detected by flow cytometry that measures the rate at which proliferating cells transit the cell cycle (i.e. cell‐cycling phenotype) was determined. This relationship between Tag concentration and phenotype was not linear. Instead, the cell‐cycling phenotype became saturated at relatively low Tag concentrations, i.e. a further increase in Tag concentration did not change the phenotype. The dose‐response relationship between Tag and a second phenotype, colony formation in soft agar, was also determined. Colony formation in soft agar is a measurement of cell transformation. In contrast to the cell‐cycling phenotype, transformation was linearly related to Tag over the entire 20‐fold Tag concentration range. This phenotype did not saturate at high Tag concentrations. Therefore, the dose‐response relationship between Tag concentration and the cell‐cycling phenotype was different from that between Tag concentration and cellular transformation. Since the Tag gene is comprised of multiple genetic domains that independently affect cellular proliferation, one possibility is that the differences in dose‐response of the two phenotypes indicate that different genetic domains of the gene are necessary for production of each phenotype.
A dual block to cell cycle progression in HL60 cells exposed to analogues of vitamin D,
Authors: Godyn J. J. , Xu H. , Zhang F. , Kolla S. , Studzinski G. P.
Published: May 2008

Abstract. The physiologically active form of vitamin D3, 1,25‐dihydroxy‐vitamin D3, (1,25(OH)2, D3), induces differentiation of several types of myeloid leukaemia cells. The acquisition of monocyte‐like phenotype is accompanied by slower progression through the cell cycle, and G1, block has been reported to be the basis of this effect. It is shown here that human promyelocytic leukaemia HL60 cells treated with analogues of vitamin D3, which are potent inducers of monocytic differentiation, have an additional cell cycle block. Exposure to 10‐7m 1,25(OH)2, D3, or 1,25‐(OH)2,‐16‐ene‐D3 resulted in monocytic differentiation and the expected G1, block evident at approximately 48 h in a rapidly differentiating variant of HL60 cells (HL60‐G), and at 96 h in the more slowly differentiating HL60‐240 cells. In addition, a G2,+M block was noted at approximately 72 h in HL60‐G and HL60‐240 cells. Exposure to vitamin D3, analogues also markedly increased the number of dikaryons, suggesting that cytokinesis was impaired more than karyokinesis. Treatment with a third analogue 25‐hydroxy‐16,23‐diene‐D3, produced little differentiation and had minimal effects on the cell cycle parameters. These findings indicate that vitamin D3, analogues regulate cell proliferation by control of the transition of G1, and G2,+M phases, reminiscent of the cdc2/CDK2 type of cell cycle control.
Leukaemic peripheral blood plasma and bone marrow plasma: comparison of influence on lymphocyte proliferation
Authors: Schultz J. C.
Published: May 2008

Abstract. Peripheral blood plasma from some children with untreated acute lymphoblastic leukaemia (ALL) exerted an inhibitory effect in vitro on phytohaemagglutinininduced lymphocyte transformation of normal peripheral blood lymphocytes. This occurred at concentrations beyond that required for optimal response as judged by reduction of blast cell formation and tritiated thymidine and tritiated uridine incorporation into DNA and RNA, respectively. In contrast, bone marrow plasma from these patients was non‐inhibitory or contained significantly less inhibitory activity. Bone marrow plasma from the majority of healthy controls was superior to their peripheral blood plasma in enhancing phytohaemagglutinin‐induced mitogenesis. The difference between an individual's bone marrow‐ and peripheral blood‐derived plasma in enhancing proliferation of patient and healthy control cells was significantly greater amongst the patients than the healthy control group; this was attributed mainly to the increased inhibitory activity of ALL peripheral blood plasma compared with normal plasma. Medium conditioned by phytohaemagglutinin‐stimulated normal peripheral blood lymphocytes was effective in neutralizing the inhibitory activity of ALL peripheral blood plasma. Taken together, these in vitro results are at least suggestive that in vivo, in healthy subjects, the rapidly proliferating cells in the bone marrow and the ‘resting’ blood cells in the circulation may be under the influence of a fine balance of different types and/or levels of humoral growth stimulatory and inhibitory factors and that in ALL an unstable balance of these factors exists. The decreased proliferation of circulating blast cells compared with bone marrow blasts in ALL may be attributed, at least in part, to exposure to the different levels of inhibitor(s) in the circulation and bone marrow as demonstrated in vitro by our results.
Analysis of growth of multicellular tumour spheroids by mathematical models
Authors: Marušić M. , Bajzer Ž. , Freyer J. P. , Vuk‐Pavlović S.
Published: May 2008

Abstract. We wished to determine the applicability of previously proposed deterministic mathematical models to description of growth of multicellular tumour spheroids. The models were placed into three general classes: empirical, functional and structural. From these classes, 17 models were applied systematically to growth curves of multicellular tumour spheroids used as paradigms of prevascular and microregional tumour growth. The spheroid growth curves were determined with uniquely high density of m'easurements and high precision. The theoretical growth curves obtained from the models were fitted by the weighted least‐squares method to the 15 measured growth curves, each corresponding to a different cell line. The classical growth models such as von Berta‐lanffy, logistic and Gompertz were considered as nested within more general models. Our results demonstrate that most models fitted the data fairly well and that criteria other than statistical had to be used for final selection. The Gompertz, the autostim‐ulation and the simple spheroid models were the most appropriate for spheroid growth in the empirical, functional and structural classes of models, respectively. We also showed that some models (e.g. logistic, von Bertalanffy) were clearly inadequate. Thus, contrary to the widely held belief, the sigmoid character of a three or more parameter growth function is not sufficient for adequate fits.
The fraction of cells in G1 with bound retinoblastoma protein increases with the duration of the cell cycle
Authors: Jonassen T. S. , Seglen P. O. , Stokke T.
Published: May 2008

Abstract. The retinoblastoma gene product (pRB) is a nuclear phosphoprotein with growth‐suppressing effects. During early G, phase, pRB is underphosphorylated and bound in the nucleus. The association between the duration of the cell cycle/G, phase and the fraction of cells in GI with bound pRB was studied in the human pre‐B cell line Reh. The cell‐cycle duration was varied by growing cells at different concentrations (25, 10,2,0.5 and 0%) of fetal calf serum (FCS); pRB binding was studied by flow cytometry. The culture doubling time increased from 21 h in 25% FCS to 54 h in 0.5% FCS. Cell death occurred in the absence of FCS, and the culture doubling time therefore could not be defined. The fraction of cells in G, did not change significantly with decreasing FCS concentration (0.47 in 25% FCS, 0.52 in 0% FCS). In contrast, the fraction of G, cells with bound pRB increased from 0.12 in 25% FCS to 0.65 in 0% FCS. Continuous labelling with bromodeoxyuridine demonstrated that the growth fraction was close to unity at all FCS concentrations down to 0.5%, hence, the duration of the cell cycle was equal to the culture doubling time under these conditions. The duration of early G, phase (where pRB is underphosphorylated and bound) increased 10‐fold, while the duration of late G, phase increased twofold, for Reh cells grown in 0.5% FCS compared with cells grown in 25% FCS. The increase in the duration of late G1, and the increased S and G,+M phase transit times, indicate that other factors, in addition to pRB kinase activity, regulate the duration of G, and the cell cycle of serum‐deprived Reh cells.
Regulation of differentiation and protein kinase C expression in 3T3 T proadipocytes: effects of TGF‐β and transformation
Authors: Sparks R. L. , Strauss E. E. , Manga A. V.
Published: May 2008

Abstract. We are studying the mechanisms that regulate proliferation and differentiation of normal 3T3 T proadipocytes and neoplastically transformed clones which have lost the ability to differentiate. The phorbol ester 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) and transforming growth factor β (TGF‐β) are known inhibitors of the same step of the differentiation process in 3T3 T cells. Here, we examined the expression of the phorbol ester receptorJprotein kinase C (PKC) during adipocytic differentiation of 3T3 T cells and its modulation by the differentiation inhibitor TGF‐β. PKC receptor assays were performed using a tritiated analogue of TPA and it was found that PKC receptor levels decreased approximately threefold during differentiation. Northern blot analyses revealed an even greater decrease of PKC transcripts during differentiation. TGF‐β inhibited not only differentiation, but the differentiation‐dependent decrease in PKC levels as well. Transformed 3T3 T cells which have lost the ability to differentiate were found to express aberrant levels of PKC. The data suggest that TGF‐β may inhibit differentiation via a PKC‐dependent pathway and that disruption of normal PKC levels or its regulation may be involved in the loss of differentiation control in transformed 3T3 T cells.
Effects of mitomycin C and porfiromycin on exponentially growing and plateau phase cultures
Authors: Rockwell S. , Hughes C. S.
Published: May 2008

Abstract. Laboratory studies and clinical trials are exploring the use of hypoxia‐directed cytotoxic agents as adjuncts to radiotherapy. Because hypoxia and the microenviron‐mental inadequacies associated with hypoxia in solid tumours inhibit cell proliferation, an essential requirement for the successful use of hypoxia‐directed drugs in cancer therapy is that these drugs be toxic to quiescent tumour cells, as well as tumour cells progressing rapidly through the cell cycle. The experiments reported here compared the cytotoxicities of mitomycin C and porfiromycin to exponentially growing and plateau phase cultures of EMT6 mouse mammary tumour cells. The proliferative status of the cultures did not influence the cytotoxicity of mitomycin C under either aerobic or hypoxic conditions, or the cytotoxicity of porfiromycin in air. Exponentially growing cultures were slightly more sensitive than plateau phase cultures to porfiromycin in hypoxia, but the difference between the sensitivities of proliferating and quiescent cells was much smaller than the difference between aerobic and hypoxic cells. No evidence for repair of potentially lethal damage was found after treatment with porfiromycin in air or in hypoxia; this is in agreement with previous findings for mitomycin C. Mitomycin C and porfiromycin therefore exhibit the toxicity to quiescent cells needed for effective use as hypoxia‐directed drugs for the treatment of solid tumours.
Changes in oncogene expression in ascite tumour cells during ageing
Authors: Biagetti M. , Fazia M. A. Della , Servillo G. , Viola‐Magni M. P.
Published: May 2008

Abstract

The expression of two oncogenes, c‐myc and c‐fos, was studied in an ascitic tumour (ATPC+) at different times after implantation. The specific mRNA synthesis was analysed by Northern blot analysis. The presence of the oncogene proteins was shown by immunofluorescence using flow cytometry and referred to the distribution of the cells in the different cell phases. The results show that both oncogenes are expressed by ATPC+ tumour cells. c‐my is expressed 5, 8 and 12 days after implantation, although with a different intensity, and the protein is mainly present in S or S+G2 phase cells. The c‐fos oncogene is expressed only 12 days after tumour implantation and the cells labelled with the specific antibody are mainly in G1 phase. We conclude that c‐myc is principally correlated with proliferative activity, whereas c‐fos is expressed by non‐cycling cells.
Evaluation of the growth rate of MCF‐7 breast cancer multicellular spheroids using three mathematical models
Authors: Olea N. , Villalobos M. , Nuñez M. I. , Elvira J. , de Almodóvar J. M. Ruiz , Pedraza V.
Published: May 2008

Abstract. Growth data on 60 multicellular spheroids of MCF‐7 human breast cancer cells were fitted, on an individual basis, by the Gompertz, Bertalanffy and logistic equations. MCF‐7 spheroids, initiated and grown in medium containing oestrogens, exhibited a growth rate that decreased continuously as spheroid size increased. Plots of spheroid volume v. time generated sigmoid curves that showed an early portion with an approximately exponential volume increase; a middle region or retardation phase characterized by a continuously decreasing growth rate; and, finally, a late segment or plateau phase approaching zero growth rate, that permitted an estimate of the maximum spheroid size (Vmax). Growth curves generated by MCF‐7 spheroids under different experimental conditions (hormones, drugs and radiation exposures) can be compared after normalization. Linearized forms of the fitted Gompertz curves provided a convenient way to express differences in growth rate.
Abnormal DNA synthesis in polyamine deficient cells revealed by bromodeoxyuridine—flow cytometry technique
Authors: Fredlund J. O. , Johansson M. , Baldetorp B. , Oredsson S. M.
Published: May 2008

Abstract. Chinese hamster ovary cells were seeded in the absence or presence of the polyamine synthesis inhibitor 2‐difluoromethylornithine (DFMO). At 14 days after seeding, the cells were labelled for 15–120 min with the thymidine analogue bromo‐deoxyuridine (BrdUrd) and they were then fixed directly after the labelling period. In addition, cells were labelled for 30 min and they were then allowed to progress in BrdUrd‐free medium during a defined post‐labelling time before fixation. An indirect immunofluorescence technique, using the monoclonal BrdUrd antibody and the intercalating stochiometric DNA stain, propidium iodide, was applied to enable quantification of cellular BrdUrd and DNA contents, respectively, by flow cytometry (FCM). By comparing the mean DNA content of BrdUrd‐labelled cells to the mean DNA contents of G1 and G2 cells, a relative measure of the position of the BrdUrd‐labelled cells was obtained (relative movement). Relative movement data, obtained from control and DFMO‐treated cells fixed directly after BrdUrd labelling, indicated that DFMO‐treated cells entered S phase at a normal rate, while their progression through S phase was impaired. DNA histograms of BrdUrd‐labelled control cells fixed directly after labelling showed that most cells were found in early and late S phase, while DNA histograms of BrdUrd‐labelled DFMO‐treated cells showed that most cells were in early S phase, indicating a delayed progression through S phase. Analysis of relative movement of cells that were allowed to progress in BrdUrd‐free medium after labelling showed that DFMO treatment resulted in a significant lengthening of the DNA synthesis time. Labelling index was significantly higher in DFMO‐treated, growth‐inhibited cells than in early plateau phase control cells indicating an S phase accumulation in the former cells.
Expression of the 170‐kDa and 180‐kDa isoforms of DNA topoisomerase II in resting and proliferating human lymphocytes
Authors: Prosperi E. , Negri C. , Marchese G. , Ricotti G. C. B. Astaldi
Published: May 2008

Abstract. The expression of the 170‐kDa (α) and the 180‐kDa (β) isoforms of DNA topoisomerase II (topo II) was investigated with two specific monoclonal antibodies (MoAbs) in human peripheral blood lymphocytes (PBL), before and after phyto‐haemoagglutinin (PHA) stimulation. Binding of each MoAb was detected by indirect immunofluorescence labelling and quantified with flow cytometry. In resting PBL, the intensity of immunostaining was very low for both isozymes; however, topo IIβ‐associated immunofluorescence was about 2.5 times significantly higher (P<0.001) than that associated with the a isoform. Between 48 and 72 h of PHA stimulation, when the highest percentage of cells in S and G2+M phases was found, the levels of topo IIα and β increased up to about 30 and 10 times the value measured in resting PBL, respectively. Thus, the two isoforms reached comparable immunofluorescence values. At longer stimulation periods (96–120 h), topo IIα immunofluorescence was not significantly changed, while that relative to topo IIβ declined to about 50% of the peak value (P<0.02). At this time however, topo IIα‐associated immunofluorescence was not significantly different from that related to the β isozyme. These results suggest that in resting PBL topo IIα is required at levels lower than topo IIβ, while in proliferating lymphocytes both isoforms are expressed to significantly higher levels.
Slowly cycling (label‐retaining) epidermal cells behave like clonogenic stem cells in vitro
Authors: Morris R. J. , Potten C. S.
Published: May 2008

Abstract. Slowly cycling label‐retaining epidermal cells were identified by light microscopic autoradiography in the dorsal epidermis and hair follicles of adult mice 8–10 weeks after twice daily injection of [3H]dT on days three through five after birth. Pulse‐labelled epidermal cells were identified in the epidermis and hair follicles of 7–8 week old mice 1 h after a single injection of [3H]dT at 8.00 a.m. For mice of both groups, epidermal cells including those from the hair follicles were harvested by trypsinization and were cultured from low density on feeder layers of irradiated Swiss mouse 3T3. On days 2, 4, 5, 7, 10 and 12, the cultures were fixed and processed for light microscopic autoradiography, and the distribution of labelled nuclei was quantified. On day 2 of culture, both label‐retaining cells (LRC) and pulse labelled cells (PLC) were found primarily as single cells. After five days, LRC were found as pairs and clusters having silver grain counts consistent with their division. In contrast, PLC remained primarily as single cells. These results suggest that LRC may divide to form colonies (are clonogenic) whereas PLC are rarely clonogenic. The significance of this experiment is that it suggests that the LRC may not only be persistent in the epidermis, but that they may also be cells with relatively greater proliferative potential than the PLC and are thus likely to be stem cells.
Proliferation and immunoglobulin secretion of lymphoblastoid cell lines are differently affected by soluble cytokines
Authors: Pistillo M. P. , Cella M. , Mantero S. , Ferrara G. B.
Published: May 2008

Abstract. The aim of our study was to investigate whether supernatant from lipopoly‐saccharide‐activated monocytes (monocyte‐factor) and/or cytokines could enhance secretion of human monoclonal antibodies specific to HLA antigens produced by Epstein—Barr virus lymphoblastoid cell lines (EBV‐LCLs). In a low cell density culture system, the monocyte‐factor significantly stimulated cell growth of three monoclonal and two polyclonal EBV‐LCLs while no enhancement of immunoglobulin production was observed. The enhancement of proliferation was completely neutralized by an antiserum to human IL‐6 suggesting that IL‐6 was required for the stimulation of growth of LCLs. The effect of cytokines on proliferation showed large variations among the cell lines, with IL‐lβ generally inducing the highest response. Of the cytokines tested, only IL‐2 was able to enhance total immunoglobulin secretion due to the induction of a higher production of light chains. The specific anti‐HLA activity was slightly increased by IL‐10 although this cytokine had no effect on total immunoglobulin concentration or proliferation.
The cell cycle effects and induction of apoptosis by 5‐bromouridine in cultures of human leukaemic MOLT‐4 and HL‐60 cell lines and mitogen‐stimulated normal lymphocytes
Authors: Li X. , Patel R. , Melamed M. R. , Darzynkiewicz Z.
Published: May 2008

Abstract. 5‐Bromouridine (BrUrd) and several analogues of this antimetabolite exhibit antiviral activity and are potent inhibitors of the human immunodeficiency virus. The antitumour activity of BrUrd, however, in comparison with 5‐fluorouridine or 5‐fluorouracil, is less pronounced. Because BrUrd is incorporated into RNA and can be detected immunocytochemically and analysed by cytometry, it may prove useful as an RNA precursor to assay cell transcriptional activity. The aim of the present study was to evaluate the cell cycle effects of BrUrd incorporation in human lymphocytic leukaemic MOLT‐4 and HL‐60 cells and mitogenically stimulated normal peripheral blood lymphocytes. BrUrd suppressed cell proliferation and induced cell death; when measured after 72 h of treatment, the LD50 was 10 and 20μm for HL‐60 and MOLT‐4, respectively, and LD90 was 100μm, for both cell lines. BrUrd was maximally incorporated by the cells progressing through S phase of the cycle and the S phase cells were most severely perturbed by the drug, which was detected in RNA but not in DNA. Suppression of the S phase traverse of MOLT‐4, HL‐60 and normal lymphocytes was seen at ≫30μm BrUrd concentration. Also sensitive was the cell traverse through G2+M which, in the case of lymphocytes, HL‐60 and MOLT‐4 cells, was perturbed at ≫30, ≫200 and 500μm BrUrd concentrations, respectively. Transition of lymphocytes from G0 to G1 was little affected at <100μm, and, although suppressed, was still observed even at 500μm BrUrd concentration. Apoptosis of HL‐60 cells and lymphocytes was observed at ≫50μm BrUrd after 24 h of incubation; death of MOLT‐4 cells had mixed features of apoptosis and necrosis and resembled the ‘mitotic’ or ‘reproductive cell death‘ as described in other cell systems. The cytostatic and cytotoxic effects of BrUrd should be taken into consideration when using this antimetabolite as an antiviral agent in the clinic or as an RNA precursor in assays of cell transcriptional activity.
Cell kinetic studies in mouse tongue mucosa by autoradiographic, immunohistochemical and flow cytometric techniques
Authors: Dörr W. , Arui J. , Beisker W. , Schultz‐Hector S.
Published: May 2008

Abstract. A number of techniques, including autoradiography after in vivo administration of tritiated thymidine ([3H]dT), immunohistochemistry after in vivo administration of bromodeoxyuridine (BrdUrd), and flow cytometry (FCM) with and without BrdUrd detection were compared in the epithelium of ventral mouse tongue. Investigation of the diurnal proliferative rhythm by immunohistochemical detection of incorporated BrdUrd with different primary antibodies in combination with the alkaline‐phosphatase‐anti‐alkaline‐phosphatase technique, the peroxidase‐anti‐perox‐idase method, and an indirect method with a polyclonal peroxidase‐conjugated secondary antibody yielded results similar to standard autoradiography. Preparation of single cell suspensions for flow cytometry was not successful. A maximum yield of about 8.5% of the original cell number was achieved by ultrasound disintegration in combination with trypsin and dithioerythrol treatment, but neither a GdG, peak nor a G2+ M peak was observed in DNA histograms. A better yield of about 38% of the original nuclei number was obtained by preparation of suspensions of nuclei using citric acid and the detergent Tween 20 in combination with magnetic stirring. Both S‐phase index and BrdUrd labelling index could be determined by FCM and showed the normal diurnal variations. However, the BrdUrd labelling index in suspensions of nuclei was significantly higher than the labelling index determined after immunohistochemistry. The FCM S‐phase index at times of day with low DNA synthesizing activity was higher than the BrdUrd index, indicating a fraction of unlabelled S‐phase cells. In conclusion, detection of incorporated BrdUrd in oral mucosa by immunohistochemical techniques or flow cytometry is feasible and provides a useful tool for fast measurements of proliferation.
Repair, redistribution and repopulation in V79 spheroids during multifraction irradiation
Authors: Brown R. C. , Durand R. E.
Published: May 2008

Abstract. Development of predictive assays for measuring tumour radiosensitivity has generated much recent interest, particularly with the recognition that tumour cell survival at doses of about 2 Gy may correlate well with tumour curability. Clinical data, however, suggest that overall treatment time may be of considerable significance in radioresponsive tumours, especially for rapidly growing tumours capable of accelerated repopulation. Because neither factor can be repeatedly assessed in human tumours, we used cells growing as multicell spheroids to determine whether the initial radiation response would be predictive for multifraction exposures, or whether other factors including repopulation rate should be considered. Potential problems of hypoxia and reoxygenation were avoided by using small spheroids which had not yet developed radiobiologically hypoxic regions. Repair and redistribution dominated the responses in the first two or three exposures, with repopulation playing a minor role. As the fractionation schedule was extended, however, repopulation between fractions largely determined the number of viable cells per spheroid. We conclude that the radiation response of cells from untreated spheroids provides a general indication of net sensitivity, but that repair and redistribution produces considerable variation in radiosensitivity throughout a fractionation protocol. Ultimately, repopulation effects may dominate the multifraction response.
Glutamic acid and tryptophan do not prevent vinblastine‐induced apoptosis in a human lymphoma cell line
Authors: Harmon B. V. , Ishii H. , Allan D. J.
Published: May 2008

Abstract. The amino acids glutamic acid and tryptophan reportedly reverse the antitumour effect of the cancer chemotherapeutic drug vinblastine. Recent studies have shown that a large part of vinblastine's antitumour effect is due to induction of apoptosis. In this morphological and DNA gel electrophoretic study, we have looked for inhibition of vinblastine‐induced apoptosis by glutamic acid and tryptophan and, for comparison, have examined their effect on apoptosis induced by another cytotoxic drug, etoposide. Inhibition tests were also performed using the protein synthesis inhibitor, cycloheximide, and the protein kinase C activator, phorbol ester (PDBu). Apoptosis induced by vinblastine and etoposide was not prevented by cycloheximide but was abrogated to some extent by PDBu. Glutamic acid and tryptophan had no effect on the level of apoptosis produced by either vinblastine or etoposide. The reason for the reported reversal of antitumour effect of vinblastine by glutamic acid and tryptophan remains unclear.
An improved mathematical method to estimate DNA synthesis time of bromodeoxyuridine‐labelled cells, using FCM‐derived data
Authors: Johansson M. C. , Baldetorp B. , Bendahl P. O. , Johansson R. , Oredsson S. M.
Published: May 2008

Abstract. Chinese hamster ovary cells in vitro were pulse‐labelled with bromodeoxyuridine (BrdUrd and were then allowed to progress through the cell cycle. Every half hour after labelling, cells were harvested and prepared for simultaneous flow cytometric determination of DNA content and incorporated BrdUrd, with the intercalating dye propidium iodide and with a monoclonal antibody against incorporated BrdUrd, respectively. The relative movement (RM), i.e. the relative mean DNA content of the moving cohort of BrdUrd‐labelled cells in relation to that of G1 and G2 cells, was calculated. RM was then used to calculate DNA synthesis time (TS), at all post‐labelling times (t). Since labelled cells in G2 and mitosis (M) in addition to S phase cells, are included in the cohort of moving labelled cells, and since the time of G2 and M (Tg2+M) phases is finite, a non‐linear relationship exists between RM and post‐labelling time. Because of this, the use of a linear formula in the calculation of TS yields results that are affected by t. We found that RM data can be corrected with regard to TG2+M resulting in the derivation of a non‐linear TS formula. This non‐linear TS formula gave results that were nearly independent of t. Moreover, windows were set in the mid DNA distributions for G1, S and G2+ M cells in the bivariate DNA v. BrdUrd cytograms, to estimate the fraction of BrdUrd‐labelled cells in each window at every post‐labelling time. Plots of the fraction of BrdUrd‐labelled cells v. post‐labelling time were then made for each window. TS obtained in this way was in agreement with TS obtained with the corrected RM method. In conclusion, we present a method to calculate Ts which theoretically first makes the determination of RM independent of TG2+M, and secondly compensates for the non‐linear function of RM with post‐labelling time caused by accumulation of BrdUrd‐labelled cells in G2+ M.
The effects of interleukin 4 on the cell cycle of a human B‐cell lymphoma line
Authors: Shubinsky G. , Schlesinger M.
Published: May 2008

Abstract. Exposure of Farage, a human B‐cell lymphoma line, to IL‐4 for 3–11 days led to inhibition of tritiated thymidine ([3H]dT) uptake by the cells. Study of the incorporation of 5‐bromodeoxyuridine by Farage cells showed that IL‐4 reduced significantly the number of cells in the S phase of the cell cycle and increased the proportion of cells in the G1 phase. Limiting dilution analysis of proliferation demonstrated that IL‐4 decreased the frequency of clone‐forming cells by 40%. IL‐4 did not reduce the viability of Farage cells. On the contrary, IL‐4 diminished the spontaneous death of Farage cells in culture, as determined by pulse chase analysis of cells which were labelled with [3H]dT. Moreover, the pre‐treatment of Farage cells with IL‐4 prevented their death induced by exposure to a high dose of staurosporine. IL‐4 abrogated the staurosporine‐induced arrest of cells in the G2+ M phase and replaced it by accumulation of cells in the G1 phase. IL‐4 protected Farage cells from the radioactive suicide caused by the uptake of [3H]dT by dividing cells. The cytokine failed to prevent the damage to Farage cells exerted by mitomycin C, which affected cellular DNA regardless of the phase of the cell cycle. The data obtained showed that IL‐4 inhibited the division of B cells by arresting their progression through the early stages of the cell cycle. This inhibition of the cell efflux from G1 phase plays an important role in the protection against cell death during further stages of the cell cycle.
Analysis of autocrine growth regulators produced by chondrogenic fibroblasts of chick embryo sclera in protein‐free primary cultures
Authors: Watanabe K. , Miyazaki T. , Oiji F. , Tsuda T.
Published: May 2008

Abstract. In the chick embryo there is a population of chondrogenic fibroblasts known as scleral fibroblasts. Scleral fibroblasts in primary culture secrete multiple autocrine growth‐promoting factors, scleral autocrine factors (SAFs), into protein‐free medium (Watanabe et al. 1989). One such factor, SAF‐IIa, which is heat‐labile and binds to heparin, shows strong DNA synthesis‐promoting activity on the mouse fibroblast cell line, BALB/c 3T3 A31 cells and has a molecular weight of c. 16 kDa by gel filtration. These data suggest that SAF‐IIa is related to growth factors of the FGF family. However, the effects of heparin augmentation on the growth‐promoting activity suggest that SAF‐IIa is not identical to aFGF or bFGF, when assayed on scleral fibroblasts and also on BALB/c 3T3 A31 cells. The other heat‐labile autocrine growth‐promoting factor, SAF‐IIb, shows weak binding to heparin and no growth‐promoting activity for BALB/c 3T3 A31 cells. The heat‐resistant growth factor, SAF‐I, is effective in enhancing the proliferation of BALB/c 3T3 cells, and its activity is increased by heat treatment. Whole‐embryo fibroblasts, which show low autocrine growth in protein‐free medium, produce mainly SAF‐IIa‐like growth‐promoting activity and do not produce SAF‐I. This indicates that the strong proliferative activity of scleral fibroblasts in vitro can be attributed to the production of a strong and stable autocrine factor, SAF‐I, in the growing phase (Watanabe et al. 1989) and this is a specialized property of the chondrogenic cells of the sclera.
Optimal conditions of fixation for immunohistochemical staining of proliferating cell nuclear antigen in tumour cells and its cell cycle related immunohistochemical expression
Authors: Wada T. , Shimabukuro T. , Matsuyama H. , Naito K. , Skog S. , Tribukait B.
Published: May 2008

Abstract. Comparison of the results of immunohistochemical expression, such as proliferating cell nuclear antigen (PCNA) in archival material of tumours, with the clinical course is extremely valuable in determining the biological malignant potential of newly detected tumours. To obtain stable and reproducible results of immunohistochemical expression of the PCNA of tumours, we studied the optimal conditions of fNation, processing and staining of samples using animal‐implanted MBT‐2 cells derived from chemical‐induced mouse bladder carcinoma and PC10, a monoclonal antibody for PCNA. The intensity of staining and PCNA positive rates were stable and reproducible when resected specimens from the tumours were covered with gauze wetted with physiological saline at room temperature before fixation for less than 12 h, fixation in formaldehyde was less than 48 h, and paraffin‐embedded sections were dried for less than 1 h. The most clear staining of PCNA positive nuclei was observed when 10% neutral buffered formaldehyde was used as a fixative. The PCNA positive rates obtained under these conditions was compared with the bromodeoxyuridine (BrdUrd) labelling indices. Although the average PCNA positive rate was significantly higher than the BrdUrd labelling index (P≪0.01), a significant correlation between PCNA positive rates and BrdUrd labelling indices was observed. In order to study the cell cycle related expression of PCNA, Ehrlich ascites tumour cells were separated by centrifugal elutriation. PCNA positive nuclei were observed in all phases of the cell cycle including G,. Occurrence of PCNA positive G1 cells was expected at a half‐life of the PCNA‐protein of 20 h and a tumour cell doubling time of about 24h. Thus, the percentage of PCNA positive nuclei in a conventionally paraffin‐embedded specimen of a tumour reflects both the growth fraction and the doubling time of the tumour and it may be a useful parameter of the biological malignant potential of tumours.
A stochastic model for dual label experiments: an analysis of the heterogeneity of S phase duration
Authors: Kroll S. , Char D. , Kaleta‐Michaels S.
Published: May 2008

We develop a statistical approach for the study of S phase duration in experiments using sequential pulses of two thymidine analogues. Cell entrance into S phase is assumed to follow a possibly nonhomogeneous Poisson process, and the duration in S phase independently follows an unspecified distribution, thus allowing the possibility of variable S phase duration times for cells. Several conclusions regarding experimental design considerations are reached. The availability of three labelled cell subgroups comprising cells receiving exactly one of the two thymidine analogues or receiving both thymidine analogues at least doubles the efficiency of the mean S phase duration estimate compared to conventional estimates based on two labelled groups. Increasing the duration between the two thymidine analogue pulses can also dramatically increase the efficiency. The modelling technique was applied to fourteen uveal melanomas from patients who received in vivo injections of two thymidine analogues, bromodeoxyuridine (BrdUrd) and iododeoxyuridine (IdUrd). Counts of labelled cells were consistent with steady‐state time homogeneous entry into S phase and nonhomogeneous spatial entry into S phase.
Effects of IL‐11 on the growth of intestinal epithelial cells in vitro
Authors: Booth C. , Potten C. S.
Published: May 2008

The network of interacting factors that control proliferation in the intestinal epithelium is largely unknown. Recently, IL‐11 was found to protect animals from lethal doses of cytotoxic agents. Part of this protective action was ascribed to a reduced level of damage in the intestinal epithelium. Whether this was due to a direct effect on epithelial cell cycle progression was unclear. We have addressed this question in vitro and found that IL‐11 reversibly inhibited proliferation in untransformed small intestinal IEC18 cells. However, IL‐11 did not inhibit transformed SW620 or HT29 colonic cell lines. IL‐6 behaved in a similar manner to IL‐11. Thus, these results suggest that IL‐11 may be an ideal therapy adjuvant, protecting normal cells and further, these results suggest that IL‐11 may be involved in the normal growth controls in the intestinal epithelium. The inhibitory response evoked by IL‐11 is lost during carcinogenic transformation.