bluuebunny/crossref_metadata_2025 huggingface dataset:
Studies of the geographic patterns of c‐myc expression in bone marrow
Authors: Banavali S. D. , Silvestril F. F. , Pavelic K. , Pavelic Z. P. , Smith P. L. , Preisler H. D.
Published: May 2008

Abstract. C‐myc expression was studied semi‐quantitatively in bone marrow biopsies obtained from normal individuals, patients with non‐malignant haematological disorders and patients with various haematological malignancies. In normal bone marrow and in the bone marrow of patients with non‐malignant haematological disorders, cells containing c‐myc protein are present in small clones (average 7 ± 2·5 cells/clone) located in the centre of the histotopographic region of the biopsy. In contrast, c‐myc‐containing cells are diffusely distributed in the bone marrow of patients with acute myelogenous leukaemia (AML). In the marrow of patients with myelodysplastic syndromes evolving to AML and in patients with AML in early relapse, the clones of cells containing c‐myc are larger than those present in normal marrows (average clone size = 17·5 ± 3·5 cells). Additionally, the proportion of the cells in normal bone marrow which express c‐myc protein is less than that present in AML marrows (23·3 ± 10·17 v. 60·2 ± 6·17) and the intensity of staining is also less. Non‐Hodgkin's lymphoma patients with bone marrow involvement had distribution of c‐myc positive cells similar to those with leukaemic infiltration.
Optimal conditions for immunohistochemical determination of the in vitro DNA synthesis labelling index with bromodeoxyuridine in head and neck cancer
Authors: Browman G. P. , Kanclerz A. , Booker L. , Daya D. , Archibald S. D. , Young J. E. M. , Goldsmith C. H.
Published: May 2008

Abstract. The in vitro DNA synthesis labelling index was assessed immunohistochemically in 24 freshly obtained specimens of head and neck cancer using bromodeoxyuridine (BrdUrd) as the DNA precursor to determine the influence of BrdUrd concentration on labelling index (LI). Initially, tumour fragments were incubated in varying concentrations of BrdUrd from 2 to 100 μM for 2 h, and BrdUrd was detected with an anti‐BrdUrd monoclonal antibody using immunoperoxidase labelling. There was a dose‐response gradient with mean LI varying from 1·6% at 2 μMm BrdUrd to 8·8% at 100μM. The concentration‐response gradient best fit a quadratic model when LI was plotted against log BrdUrd concentration (r= 0·65, P < 0·0001).

Eleven additional tumours were then studied to determine whether LI increased for BrdUrd concentrations above 100 μM. The mean LI at 125 μM and at 150 μM in these 11 tumours did not differ from the value at 100 μMm, suggesting a plateau at this level. The gradient effect accounted for 17% of the variance in LI, while 60% of the variance was explained by between tumour differences. Within individual tumours, three response patterns were observed: (i) LI rose at a constant rate to the highest concentration tested (n = 8), (ii) the LI plateaued or declined at high BrdUrd concentrations (n= 6); and (iii) there was a biphasic slope slope in which the rate of rise in the LI increased at the higher BrdUrd concentrations (n = 2).

The data show that BrdUrd concentration is an important variable in the immunohistochemical assessment of the in vitro LI in head and neck cancer.
The decay of autoradiographic grain number over crypt base columnar cells in murine ileum as a measure of their generation time
Authors: Chwalinski S. , Potten C. S.
Published: May 2008

Abstract. The decay in the number of grains over [3H]‐thymidine labelled crypt base columnar cells (BCC) in autoradiographs of the ileum of BDFl mice has been studied. The results revealed that using the conventional grain count halving (GCH) method it is possible to obtain an estimation of the generation time (Tc) of the proliferative BCC cells in the Paneth cell zone (PC‐zone) of 18.8 ± 0.74 h. This lies within the range obtained by the percent labelled mitoses (PLM) method, but is shorter than most values obtained by stathmokinetic methods. The present data show no evidence for a shortening of the cell cycle 3 days after irradiation (8 Gy) which is contrary to some earlier observations. Some reasons for this discrepancy are discussed. The comparatively high labelling index of the BCC allows a larger amount of data to be easily collected, compared with the PLM technique, and correction factors which take into account the complicated shape of the bottom of the crypt are not required.
A cytokinetic approach to determine the range of O2‐dependence of pyrimidine(deoxy)nucleotide biosynthesis relevant for cell proliferation
Authors: Löffler M.
Published: May 2008

Abstract. In vitro cultured Ehrlich ascites tumour (EAT) cells were used because of the ease of their manipulation under different levels of hypoxia. They were used to clarify further the complex mechanism of oxygen‐dependent cell proliferation. On reducing the oxygen concentration from 20% to lower levels (1–7%) an increase in the length of the population doubling time with concomitant reductions in protein, RNA and DNA content of cultures were observed. The incorporation of [14C]HCO3‐‐into the RNA fraction of cells by de novo biosynthesis of uridine monophosphate (UMP) was reduced proportionally to the microenvironmental O2 tension. Uptake of this labelled precursor by cells in the presence of N‐phosphonoacetyl‐L‐aspartate was found to be similarly inhibited. To correlate the reduction of cell growth under hypoxia with the functional pyrimidine supply, hypoxic cells were cultured in the presence of a balanced mixture of deoxynucleosides and/or uridine (100 μm deoxycyti‐dine, 10 μ deoxyadenosine, 10 μ deoxyguanosine, 100 μM uridine). Above 3% O2 in the protective atmosphere, no improvement of growth parameters by the exogenous pyrimidinenucleotide precursors was obtained, whereas these compounds had a positive influence below this level. The increase in cell number was raised to about 60% of that of control cultures (20% O2) irrespective of the oxygen tension. In addition, when above 3% O2 the incorporation of HCO3‐‐ into RNA was comparable to that of controls, indicating that the pyrimidine de novo pathway is not a limiting factor in RNA biosynthesis. In conclusion, whereas at suboptimal O2 levels (5–7%) no correlation between pyrimidine metabolism and reduction of proliferation rate appears to exist, at low O2 concentrations (<3%) the rate of orotate/UMP production seems to be an important factor in the growth cessation of EAT cells; at critical O2 tensions (<1%) the lack of pyrimidine‐deoxynucleosides substantially reduces cell cycle progression.
Clonal heterogeneity in specific growth rate of Succhuromyces cerevisiue cells
Authors: Wheals A. E. , Lord P. G.
Published: May 2008

Abstract. The cell volume increase in individual clones of cells of the yeast Succhuromyces cerevisiue has been measured using time lapse cinematography in populations showing steady state balanced exponential growth. There were significant differences in clonal specific growth rates within the population in each of 10 experiments using different strains on different media supporting different growth rates. The results suggest that specific growth rates of cells which are either genetically identical or very closely related can be different and this difference can be propagated over at least three generations. Since the proliferation rate in yeast is determined by growth rate, these observed differences provide an additional source of cell cycle variability for yeast cells that has not been considered before. The implications for the theoretical analysis of cell cycle kinetics are examined.
Interferon affects cell growth progression by modulating DNA polymerases activity
Authors: Cataldi A. , Santavenere E. , Vitale M. , Trubiani O. , Lisio R. , Tulipano G. , Di Domenicantonio L. , Zamai L. , Miscia S.
Published: May 2008

Abstract. A multiparametric analysis of the effects of human recombinant interferon alpha type A on Daudi cells involving flow cytometry and in vitro analysis of alpha and beta DNA polymerase activities has been performed. Results have disclosed (within 60 min of interferon treatment) a decrease of alpha polymerase driven DNA synthesis persisting to at least 24 h, while beta polymerase was poorly affected. Moreover, after 24 h of interferon treatment, a reduction of BrdUrd incorporation per cell, assessed by flow cytometry, was observed suggesting that DNA synthesis in S phase cells is almost completely abolished. The analysis of the effect of interferon on the distribution of cell cycle phases indicated that the G1/S transition is not inhibited by the treatment. These results support the hypothesis that interferon generates a transient initiating signal which quickly reaches the nucleus and produces a rapid inhibition of alpha polymerase activity, leading finally to the slowing of cell cycle progression.
Cell cycle dependent expression and stability of the nuclear protein detected by Ki‐67 antibody in HL‐60 cells
Authors: Bruno S. , Darzynkiewicz Z.
Published: May 2008

Abstract. The expression and stability of the proliferation‐associated nuclear antigen detected by Ki‐67 antibody have been investigated in human promyelocytic leukaemic HL‐60 cells in relation to their progression through the cell cycle. Expression of this antigen was minimal in late G1 and early S phase cells. The antigen accumulated in the cells predominantly during S phase, and its rate of increase per cell accelerated during the second half of this phase. The accumulation of Ki‐67 antigen during S exceeded the increase in DNA content, and thus the Ki‐67/DNA ratio rose 80% from late G1 to G2+ M. This antigen rapidly disappeared from post‐mitotic cells. The half‐life of this protein estimated in post‐mitotic cells during stathmokinesis induced by vinblastine appeared to be shorter than 1 h. This rapid turnover should be compared with the relatively long (6–8 h) duration of G1 of the studied cells. In cells in which de novo protein synthesis was inhibited by 0.1 μg/ml cycloheximide, the half‐life of the Ki‐67 antigen was also found to be about 1 h regardless of the cell position in the cell cycle. Thus, the data suggest that variations in the level of this protein during the cell cycle are a consequence of its different synthesis rate rather than phase‐specific changes in the rate of its degradation. Because the late G1 and very early S phase cells express the antigen at levels only slightly above background, it is possible that, when using Ki‐67 antibody as a marker of the cell growth fraction, some late G1 cells can be erroneously classified as non‐cycling cells.
Cell proliferation in the small intestine and colon of intravenously fed rats: effects of urogastrone‐epidermal growth factor
Authors: Goodlad R. A. , Lee C. Y. , Wright N. A.
Published: May 2008

Abstract. There is marked intestinal hypoplasia in the intestine of intravenously fed (TPN) rats. Recombinant urogastrone‐epidermal growth factor (URO‐EGF) reversed these changes by significantly increasing the length of the intestinal crypts. Crypt diameter, however, was not affected to the same extent. Few differences in labelling indices were seen between the orally fed and TPN groups, however, this was the consequence of the concomitant changes in crypt population.

The number of mitoses and labelled cells per crypt, and thus the crypt cell production rates, were significantly decreased in the TPN group when compared to the orally fed. URO‐EGF significantly increased both proliferative indices and the number of dividing cells per crypt. Crypt cell production in the small intestine was restored to those levels seen in the orally fed rats, moreover, labelling per crypt in the colon was increased to more than twice that of orally fed rats. The location of the mean labelling position and the half maximum labelling position followed the changes in crypt length in the small intestine, but to a lesser extent; thus the growth fraction was significantly increased in the TPN rats in comparison with the orally fed and the URO‐EGF treated groups. Similar changes in these positions were seen in the colon, but the growth fraction was much reduced in the URO‐EGF treated rats, as a consequence of the large increase in crypt length without a concomitant alteration in label distribution.
Cell cycle analysis of asexual stages of erythrocytic malaria parasites
Authors: Jacobberger J. W. , Horan P. K. , Hare J. D.
Published: May 2008

Abstract. Intra‐erythrocytic Plasmodium species can be stained with the DNA binding dye, Hoechst 33342, and the distribution of DNA content determined for parasite populations by flow cytometric measurement of fluorescence. Analysis of this distribution will determine the parasitaemia (percentage of erythrocytes infected), and the percentages of trophozoite infected red blood cells, polyparasitized (trophozoite) red blood cells, and schizont/segmenter infected red blood cells. This analysis is based on the hypothesis that the asexual parasites cycle with single G1 period, and effectively, a single S phase with no significant G2/M period except at schizogony when the genome DNA content is equivalent to 8 N or higher, dependent on the species. Data are presented to support this model.
Apoptotic cell death triggered by camptothecin or teniposide. The cell cycle specificity and effects of ionizing radiation
Authors: Bino G. Del , Bruno S. , Yi P. N. , Darzynkiewicz Z.
Published: May 2008

Abstract. We have previously observed that the DNA topoisomerase I inhibitor camptothecin (CAM), or DNA topoisomerase II inhibitors teniposide (TEN) and amsacrine (m‐AMSA) trigger endonucleolytic activity in myelogenous (HL‐60 or KGl), but not lymphocytic (MOLT‐4) leukaemic cell lines. DNA degradation and other signs of apoptotic death were seen as early as 2–4 h after cell exposure to these inhibitors. Cells replicating DNA (S phase) were selectively sensitive whereas cells in G1 were resistant; the sensitivity of G2 or M cells could not be assessed in these studies. The present studies were aimed at revealing whether DNA repair replication induced by ionizing radiation can sensitize the cells, and to probe the sensitivity of cells arrested in G2 or M, to these inhibitors. The data show that γ‐irradiation (0.5–15 Gy) of HL‐60 cells does not alter their pattern of sensitivity, i.e. G1 cells, although engaged in DNA repair replication, still remain resistant to CAM compared with the S phase cells. Likewise, irradiation of MOLT‐4 cells also does not render them sensitive to either CAM or TEN, regardless of their position in the cell cycle. Irradiation, however, by slowing the rate of cell progression through S, increased the proportion of S phase cells, and thus made the whole cell population more sensitive to CAM. HL‐60 cells arrested in G2 either by irradiation or treatments with Hoechst 33342 or doxorubicin appear to be more resistant to CAM relative to S phase cells. Also resistant are cells arrested in M by vinblastine. The data suggest that some factor(s) exist exclusively in S phase cells, which precondition them to respond to the inhibitors of DNA topoisomerases by rapid activation of endogenous nuclease(s) and subsequent death by apoptosis. HL‐60 cells in G1, G2 or M, or MOLT‐4 cells, regardless of the phase of the cycle, appear to be protected from such a mechanism, and even induction of DNA repair replication cannot initiate DNA degradation in response to DNA topoisomerase inhibitors. These data, together with the evidence in the literature that topoisomerase I may be involved in DNA repair, suggest that a combination of these inhibitors with treatments that synchronize cells in the S phase and/or recruit quiescent cells to proliferation, including radiation, may be of value in the clinic.
Haemopoietic long‐term bone marrow cultures from adult mice show osteogenic capacity in vitro on 3–dimensional collagen sponges
Authors: Schoeters G. , Leppens H. , Gorp U. Van , Heuvel R. Van Den
Published: May 2008

Abstract. Adult murine bone marrow cells, cultured under conditions for long‐term haemopoietic marrow cultures, produce bone matrix proteins and mineralized tissue in vitro, but only after the adherent stromal cells were loaded on a 3‐dimensional collagen sponge. Provided more than 8 × 106 cells are loaded, mineralization as measured by 85Sr uptake from the culture medium, occurred in this 3‐dimensional configuration (3‐D) within 6 days. In contrast if undisrupted marrow fragments (containing more than 107 cells) are placed directly on a collagen sponge, then it requires more than 10 days before significant mineralization can similarly be detected. The 2‐dimensional (2‐D) long‐term marrow culture system allows prior expansion of the stromal cells and some differentiation in an osteogenic direction within the adherent stromal layer. This is suggested by the presence of type I collagen and alkaline phos‐phatase positive cells. However, synthesis of osteonectin and a bone specific protein, osteocalcin, as well as calcification are only observed in 3‐D cultures. Electron microscopy demonstrated hydroxyapatite mineral on collagen fibres, osteoblast‐like cells, fibroblasts, cells which accumulated lipids, and macrophages which were retained on the collagen matrices. Irradiation of confluent long‐term bone marrow cultures, prior to their loading on the collagen sponge showed that haemopoietic stem cells are not necessary for the mineralization.
Generation‐dependent control mechanisms in cell proliferation and differentiation—the power of two
Authors: Meinzer H. P. , Sandblad B. , Baur H. J.
Published: May 2008

Abstract. There is evidence that the proliferation of cells is controlled by the number of divisions after leaving a multi‐potent (stem) cell. A detailed study of the growth of tissue in the small intestinal tract, more precisely the growth of crypts and villi, suggests that not only the proliferation but also the differentiation of cells obey the same biological law. We postulate a theory of a cellular internal control mechanism: the cell‐generation control of differentiation and proliferation. This basic mechanism, together with extemal influences, determines the kinetic behaviour of the crypt‐villus system.
Fibroblastic cell cycling in collagen gels
Authors: Souren J. E. M. , Wijk R. van
Published: May 2008

Abstract. Quiescent C3H10T1/2 mouse fibroblasts resume DNA synthesis and proliferation following incubation in medium containing fresh serum both when grown in monolayer and when grown in a collagen matrix. We observed that the rate of DNA synthesis is reduced at high initial cell densities and low initial collagen concentrations. In a collagen matrix, fibroblasts contract the matrix causing an increase in cell density and collagen concentration. We studied the chronological relationship between the kinetics of DNA synthesis and the collagen matrix contraction. The rate of collagen collection per cell changes in time, dependent on initial cell and collagen concentration. The kinetics of the collagen collection showed a positive correlation with the kinetics of DNA synthesis, 16 h later.
The influence of cell co‐operation, nutrients and surface area on cell division
Authors: Blomquist E. , Brunk U. , Macieira‐Coelho A.
Published: May 2008

Abstract. Two opposite views have been proposed to explain the decline of the growth potential in cell populations with a limited life span: 1 variations in the probability of cycling and in cycling times or 2 a progressive increase in the nondividing cell fraction. Human brain‐derived cells were studied with respect to their proliferative potential under the influence of different growth conditions, using haptotactic palladium islands on agarose. The results emphasize the need for cell co‐operation, surface area and nutrients for cell division. These parameters also influence the final cell density. The results illustrate the multiple factors that can vary the probability of initiating the division cycle and stress the uncertainty of defining the irreversible non‐dividing state.
The in vitro response of human tumour cells to desferrioxamine is growth medium dependent
Authors: Voest E. E. , Rooth H. , Neijt J. P. , Van Asbeck B. S. , Marx J. J. M.
Published: May 2008

Abstract. Iron chelating agents have been demonstrated to inhibit tumour cell growth. However, in vitro and in vivo results using desferrioxamine a hexadentate iron chelating agent, for anti‐cancer treatment are not always in agreement. Therefore, we have studied the response of three human tumour cell lines (HL‐60 promyelocytic leukaemia, MCF‐7 breast cancer and HepG2 hepatoma), grown in culture medium supplemented with either human pooled (HPS) or fet al bovine serum (FBS), to desferrioxamine. Desferrioxamine, at micromolar concentrations, induced severe cytotoxicity in all tumour cell lines grown in FBS medium. When grown in HPS medium, comparable desferrioxamine cytotoxicity was observed in the millimolar range. The addition of 50% saturated human transferrin to FBS medium resulted in protection against desferrioxamine cytotoxicity. HL‐60 cells were further studied for iron metabolism characteristics. HL‐60 cells, grown in medium with FBS, were found to have an 8.4 fold increase in surface transferrin receptor (TfR) expression (P < 0.001) as compared with HL‐60 cells grown in medium with HPS. However, iron uptake of HPS cultured HL‐60 cells, after incubation with saturated human transferrin, was higher, resulting in a higher concentration of iron in HPS cultured HL‐60 cells as compared with FBS cultured cells (1.72 ± 0.02 μmol/g protein v. 1.32 ± 0.14 μmol/g protein; P < 0.001). Using desferrioxamine it was shown that TfR expression is dependent on the biological availability of iron in the cell. Consistent with the lower iron content in FBS cultured cells, we conclude that the cytotoxicity of desferrioxamine is dependent on the ability of cells to replenish cellular iron stores from the culture medium. Cells grown in FBS medium lack this ability and are therefore more susceptible to desferrioxamine.
In vitro BrdUrd incorporation of colorectal tumour tissue
Authors: Williamson K. , Halliday I. , Hamilton P. , Ruddell J. , Varma M. , Maxwell P. , Crockard A. , Rowlands B.
Published: May 2008

Abstract. This study describes a novel in vitro method for the incorporation of the thymidine analogue, bromodeoxyuridine (BrdUrd), in fresh colorectal tumour tissue. Disaggregation by pronase, collagenase and DNAse resulted in high cell yields of viable single cell suspensions, representative of the original tumour, which could be infiltrated with BrdUrd. A modified ELISA identified optimal incubation times and BrdUrd concentrations. This technique has been used in preliminary studies to investigate two important areas intrinsic in the analysis of BrdUrd colorectal cell proliferation data: 1 to determine the effects of the individual constituents of the cell culture media, in particular glutamine, on BrdUrd incorporation in suspensions of colorectal cells and 2 to examine the denaturation step. This method will have wide applicability in investigations of cell proliferation status in both normal and diseased tissue.
Heterogeneity in the mouse epidermal cell cycle analysed by computer simulations
Authors: Aarnæs E. , Clausen O. P. F. , Kirkhus B. , De Angelis P.
Published: May 2008

Abstract. Different sets of cell kinetic data obtained over many years from hairless mouse epidermis have been simulated by a mathematical model including circadian variations. Simulating several independent sets of data with the same mathematical model strengthens the validity of the results obtained. The data simulated in this investigation were all obtained with the experimental system in a state of natural synchrony. The data include cell cycle phase distributions measured by DNA flow cytometry of isolated epidermal basal cells, fractions of tritiated thymidine ([3H]TdR) labelled cells within the cell cycle phases measured by cell sorting at intervals after [3H]TdR pulse labelling, bivariate bromodeoxyuridine (BrdUrd)/DNA data from epidermal basal cells isolated at intervals after pulse labelling with BrdUrd, mitotic rate and per cent labelled mitosis (PLM) data from histologic sections. The following main new findings were made from the simulations: the second PLM peak observed at about 35 h after pulse labelling is hardly influenced by circadian variations; the peak is mainly determined by persisting synchrony of a rapidly cycling population with a G1‐duration (TG1) of 20 h to 30 h; and there is a highly significant population of slowly cycling G1‐cells (G1σ). However, no significant circadian variations were found in the number of these cells.
The control of DNA replication in hybrids between neutrophils and fibroblasts
Authors: Prudovsky I. A. , Kosimov R. B. , Sukharev S. I. , Pospelova T. V. , Prasolov V. S. , Zelenin A. V.
Published: May 2008

Abstract. DNA synthesis regulation in heterokaryons between mouse neutrophils and cultured cells of various proliferative potentials has been studied. The following features have been found. Both immortalized and non‐immortalized cells can reactivate DNA synthesis in neutrophil nuclei. The reactivation ability of cultured cells increases after immortalization and is not changed by further transformation. Neutrophils inhibit the entry of cultured cell nuclei into S phase and have no effect on ongoing DNA synthesis. Malignant cells are much less sensitive to the inhibitory action of neutrophils than non‐malignant ones. Non‐malignant immortalized cells are as sensitive to this effect as non‐immortalized cells. Neutrophil karyoplasts do not influence DNA synthesis in partner cultured cell nuclei. Cycloheximide pretreatment of neutrophils drastically diminishes their inhibitory effect.
The distribution of colony‐forming cells in the kidney
Authors: Jen Y. M. , Hendry J. H.
Published: May 2008

Abstract. A method is described for producing outgrowths of small nephron segments (average 24 cells) in culture. The method was used to estimate an overall colony‐forming efficiency of 4.6% for cells constituting the segments. Efficiency was found to be lower for thick segments (1%) than for thin segments (6%) from Henle's loop. The latter higher level indicates that precursor cells are concentrated near the middle of the nephron. For comparison, a two‐dose irradiation technique was used to calculate a mean number of 5 ± 2 (SE) clonogens per segment producing outgrowths. This tended to be higher than the value of about 1 calculated from the 65% of segments producing outgrowths, as expected if the remaining segments contained no clonogens.
Stimulator of proliferation of spleen colony‐forming cells in acute sterile inflammation
Authors: Milenković P. , Ivanović Z. , Lukić M. L. , Kataranovski M. , Lord B. I.
Published: May 2008

The presence and activity of a spleen colony‐forming cell (CFU‐S) proliferation stimulator was investigated in rat bone marrow after induction of sterile inflammation. Wistar rats were treated intraperitoneally with two 15 ml injections of 3.5% polyvinylpyrrolidone (PVP) at 18 h intervals, and the presence of CFU‐S proliferation stimulator determined in bone marrow 6, 20 and 24 h after the first injection. The marrow of these mice was used to condition medium which was then fractionated using Amicon Diaflo ultrafiltration membranes. The 30–50 kDa fraction, taken from 20 h post‐PVP‐bone marrow extract, was found to induce cycling of d8‐CFU‐S in normal mouse bone marrow. This activity was not related to the presence of interleukins‐1, ‐2 or ‐6 like activities in the material tested. The results demonstrate the existence of CFU‐S proliferation stimulator in the bone marrow of rats with sterile inflammation (i.e. in an in vivo tissue response to non‐specific cell stimulation), similar to that originally described as a macrophage product in mouse bone marrow after treatment with a variety of cytotoxic agents.
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

Quantitative 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

The 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.
THE 1993 LEWIS SCHIFFER MEMORIAL LECTURE TO THE CELL KINETICS SOCIETY
Authors: McCulloch E. A.
Published: May 2008

Abstract. Normal haemopoiesis is a cellular hierarchy headed by pluripotent stem cells capable of both self renewal and, after determination, the generation of differentiating lineages that end in terminal functional cells. The role of stem cells is crucial because only these have the capacity to generate clonal populations during development or after injury. During clonal expansion the cells are affected by many sets of receptors and ligands. These belong to at least two classes: one consists of growth factors that bind cell surface receptors and initiate signalling events; the other class contains receptors which act as ligand‐dependent transcription factors such as the intracellular steroid super‐family. In spite of this elaborate regulatory apparatus, control during clonal expansion is lax, perhaps stochastic, as evident from the great heterogeneity disclosed by examining the cellular compositions of haemopoietic clones. It may be that the large number of signals impinging on binary possible outcomes (for example self‐renewal or determination) serve to set probabilities rather than to determine outcomes. In leukaemia, many of the features of normal haemopoiesis are retained. The disease begins as transformations in normal stem cells; after additional leukaemogenic events clonal expansion yields malignant populations which are clonal in each affected individual. These dominant clonal populations retain the hierarchical organization found in the normal, the major difference is that post‐deterministic divisions in leukaemia yield descendants that retain primitive (blast) morphology although proliferative capacity is lost. In acute myeloblastic leukaemia (AML) cell culture methods are available that permit the measurement of clonogenic blast stem cells. These methods have shown that regulatory mechanisms active in normal haemopoiesis are retained in AML, including lax regulation during clonal expansion. The biological features of blast stems cells displayed by the culture technique reflect in part, events in vivo, as associations have been found between results in cell culture and clinical outcome. Thus, study of leukaemic populations provides a challenge for basic science and an opportunity for successful application in control of disease.
In vitro tumour cell growth inhibition: a comparative study between allosensitized cytotoxic T lymphocytes and lymphokine activated killer cells
Authors: Fuggetta M. P. , Alvino E. , Pepponi R. , De Filippi R. , Marini S. , Bonmassar E.
Published: May 2008

There is general agreement that several distinct subpopulations of lymphocytes, including major histocompatibility complex (MHC)‐restricted T lymphocytes and non‐restricted natural killer, or lymphokine‐activated killer (LAK), cells are active in lysing neoplastic cells. In this study experiments were designed to compare the inhibitory effects of LAK cells and allosensitized cytotoxic T lymphocytes (CTL) on in vitro growth of an Epstein–Barr virus‐transformed B‐cell line (BSM) and of a HTLV‐I producer T‐cell line (MT‐2). It was found that allosensitized CTL are more efficient at inducing BSM, or MT‐2, cell growth inhibition than LAK cells. These results are consistent with the hypothesis that MHC‐restricted T effector cells could mediate higher tumour suppressive effects than non‐MHC restricted LAK cells.
Proliferation‐differentiation pathways of murine haemopoiesis: correlation of lineage fluxes
Authors: Novak J. P. , Necas E.
Published: May 2008

Abstract. A quantitative analysis of literature data on muiine bone marrow haemopoiesis was performed in order to determine how the haemolpoietic system fulfils requirements of cell turnover in the normal steady state. In particular, the production rates (fluxes) of erythrocytes, megakaryocytes, monocytes, neutrophils, eosinophils, basophils and mast cells have been calculated and normalized. Subsequently, based on available data on relationships of various lineages, a working model of the developmental schema of haemopoiesis was derived. Since, according to the principle of conservation, the effluxes of all lineages from bone marrow in the steady state must be equal to effluxes from the compartments of respective precursors divided by the coefficient of multiplication, the correlation of lineage fluxes was used to determine the position of branching points. It was concluded that all the quantita.tive data on lineage proliferation in the normal steady state in vivo, available for analysis, are fully consistent with a simple binary model of the sequential type, similar to that suggested by Brown and coworkers (Brown G., Jones NA, Bunce CM, Owen PJ, IPatton WN. (1988) Haemopoiesis: a lottery or determinism?Differentiation, 39, 83).
Reduced variance of bone‐marrow transit time of granulopoiesis—a possible pathomechanism of human cyclic neutropenia
Authors: Schmitz S. , Franke H. , Loeffler M. , Wichmann H. E. , Diehl V.
Published: May 2008

Abstract. Human cyclic neutropenia (CN) is a haematological disorder characterized by oscillations in the numbers of neutrophilic granulocytes and other blood cells with a stable period of approximately 21 days. In most cases the neutrophils oscillate well below normal values such that these patients are chronically neutropenic. A comprehensive concept of the origin of CN is proposed. It assumes an abnormally small variance of the transit time of bone marrow cells (compared to normal human granulopoiesis) for the origin of the characteristic cycles. Furthermore, a reduced responsiveness of the immature granulopoietic bone marrow cells to the mitotic feedback stimuli is assumed to account for the subnormal neutrophil peaks. Together with feedback control provided in a simulation model of normal human granulopoiesis these two abnormalities can explain experimental and clinical cell kinetic data for bone marrow and blood in CN.
The role of a 70 kDa nuclear protein in fetal haemoglobin synthesis in K562 cells
Authors: Baliga B. S. , Shah A. K. , Yang Y.‐M.
Published: May 2008

Abstract. Previously, we reported that a 70 kDa nuclear protein may regulate fetal haemoglobin gene expression in haemin treated K562 cells. To obtain further evidence of the specific role of this 70 kDa nuclear protein, we compared the nuclear fractions isolated from phenylacetate, hydroxyurea and haemin treated K562 cells. Both phenylacetate and hydroxyurea have been used to induce fetal haemoglobin synthesis in K562 cells. Cell growth was measured by biochemical events including DNA, RNA and protein synthesis. Differentiation of K562 cells was determined by both [3H]‐leucine incorporation into fetal haemoglobin and scoring benzidine‐stained positive cells. Unlike the haemin treated cells, phenylacetate and hydroxyurea induced growth arrest and increased fetal haemoglobin gene expression in K562 cells. After four days of treatment with phenylacetate and hydroxyurea more than 50% of the cells stained positive with benzidine. The SDS‐Polyacrylamide gel electrophoretic analysis of nuclear proteins isolated from phenylacetate and hydroxyurea treated K562 cells showed that the 70 kDa protein was reduced in nuclear protein extract in both groups similar to haemin treated cells. These results suggest that the loss of the 70 kDa protein from a nuclear protein extract is not restricted to only haemin treated cells but also occurs in hydroxyurea and phenylacetate treated cells. Our results provide further evidence that the 70 kDa nuclear protein may be involved in regulating fetal haemoglobin expression through a negative control mechanism.
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.
The progression of 1,2‐dimethylhydrazine‐induced colon tumourigenesis stimulates growth and cell proliferation in the colon of the host mouse
Authors: Carter J. W. , Hardman W. E. , Cameron I. L. , Lancaster H. K.
Published: May 2008

Abstract. The aim of this study was to characterize tissue responses attributable to the stage of tumourigenesis in CF‐1 mice. The study involved initiating colon carcinogenesis by eight weekly subcutaneous injections of 1,2‐dimethylhydrazine (DMH at 12mg/kg body weight), while the control mice received injections of the carrier vehicle. All mice were killed for examination of tissue responses at 24 weeks after the completion of the injections. Each mouse was classified into one of four groups based on the histology of the most advanced colonic lesion. The four groups or stages were: 1 no lesions (non‐DMH‐treated mice); 2 mice with aberrant crypt foci (ACF); 3 mice with one or more adenomas (AD) and 4 mice with an adenocarcinoma (AC). Significant tissue specific responses were identified and related to the stage of tumourigenesis. For example, mice with AC demonstrated a stage specific and marked hypertrophy of the entire colon and spleen, while demonstrating loss of body weight with no change in weight of the thymus or liver. The colonic crypts from these AC‐bearing mice demonstrated hyperproliferation and an upward shift of the proliferative zone, with a concurrent loss of iron, but not of calcium, copper, magnesium or zinc in the liver. Splenomegaly was attributed to transition of the tumour to an invasive state. It is proposed that the AC produces a blood‐borne trophic factor which helps explain a field effect on the colonic epithelium far removed from the growing AC. This field effect can help explain how biopsy of the large bowel (usually taken from the rectum in humans) can provide morphokinetic information predictive of the stage of colon carcinogenesis.
Cell genealogies in a plant meristem deduced with the aid of a ‘bootstrap’ L‐system
Authors: Lück J. , Barlow P. W. , Lück H. B.
Published: May 2008

Abstract. The primary root meristem of maize (Zea mays L.) contains longitudinal files of cells arranged in groups of familial descent (sisters, cousins, etc.). These groups, or packets, show ordered sequences of cell division, which are transverse with respect to the apico‐basal axis of the root. The sequences have been analysed in three zones of the meristem during the course of the first four cell generations following germination. In this period, the number of cells in the packets increases from one to 16. Theoretically, there are 48 possible division pathways that lead to the eight‐cell stage, and nearly 2‐ × 106 that lead to the 16‐cell stage. However, analysis shows that only a few of all the possible pathways are used in any particular zone of the root. This restriction of pathways results from inherited sequences of asymmetric cell divisions, which lead to sister cells of unequal length. All possible division pathways can be generated by deterministic ‘bootstrap’ L‐systems which assign different life spans to sister cells of successive generations and hence specify their subsequent sequence of divisions. These systems simulate propagating patterns of cell divisions, which agree with those actually found within the growing packets that comprise the root meristem. The patterns of division are specific to cells originating in various regions of the meristem of the germinating root. The importance of such systems is that they simulate patterns of cellular proliferation where there is ancestral dependency. They can therefore be applied in other growing and proliferating systems where this is suspected.
A possible interpretation of some colony forming assays: ‘developmental tree’ model
Authors: Novak J. P. , Stewart C. C.
Published: May 2008

Abstract. The characteristic form of the probability distributions of in vitro grown colonies is analysed and experimental results are shown to be well approximated by an exponential dependence. It is argued that this type of dependence is very frequent in nature and does not provide sufficient evidence to resolve the dilemma whether the development of haemopoietic lineages proceeds according to a schema of ‘progressive and stochastic restriction’ or more limiting ‘developmental tree’ with some random control process involved. Detailed analysis of the experimental conditions and results is needed to provide insight into collective phenomena in cellular proliferation and differentiation. At present, the complexity of the phenomena involved prevents unique interpretation of the experimental distributions. Nevertheless, one particular quantitative model, based on a developmental tree concept, yields a set of the theoretical probability distributions, which are in a very good agreement with the experimental results.
A model for regulation of the cell cycle incorporating cyclin A, cyclin B and their complexes
Authors: Obeyesekere M. N. , Tucker S. L. , Zimmerman S. O.
Published: May 2008

Abstract. t. A mathematical model for the cell cycle is proposed that incorporates the known biochemical reactions involving both cyclin A and cyclin B, the interactions of these cyclins with cdc2 and cdk2, and the controlling effects of cdc25 and weel. The model also postulates the existence of an as yet unknown phosphatase involved in the formation of maturation promoting factor. The model produces solutions that agree qualitatively with a wide variety of experimentally observed cell‐cycle behavior. Conditions under which the model could explain the initial rapid divisions of embryonic cells and the transition to the slower somatic cell cycle are also discussed.
Origin and evolution of binucleate cells in cultures of HEp‐2 cells
Authors: Serrano A. , Rodilla V. , Pellicer J. A. , Pertusa J.
Published: May 2008

Abstract. The origin and evolution of binucleate cells in cultures of HEp‐2 cells have been studied by means of interval photography and time‐lapse video‐recording. Binuc leate cells most frequently formed by the fusion of two sister cells born in a previous mitosis. The study of binucleate cells has shown that they are a cellular type able to successfully undergo mitosis. However, the mitosis may be bipolar, tripolar or multi‐polar. The daughter cells arising from these divisions do not follow a clear pattern in the number of nuclei they have, instead showing a wide range of possibilities.