bluuebunny/crossref_metadata_2025 huggingface dataset:
A mathematical model of erythropoiesis in mice and rats Part 2: Stimulated erythropoiesis
Authors: Wichmann H. E. , Loeffler M. , Pantel K. , Wulff H.
Published: May 2008

Abstract A mathematical model of erythropoietic cell production and its regulation process has been proposed in a preceding paper. It is primarily based on the assumption that the number of cell divisions taking place in the CFU‐E and erythropoietic precursor stages is regulated depending on the oxygen supply of the tissue. Quantitative dose‐response relationships for in vivo erythropoiesis are suggested. Here, we demonstrate that this model adequately reproduces data obtained in situations of stimulated erythropoiesis in mice and rats. In detail, this implies a quantitative description of the following processes: (1) Changes in tissue oxygen tension (PtO2) following removal of red cells (bleeding, haemolytic anaemia) or increase in plasma volume (dilution anaemia) or decrease in atmospheric oxygen pressure (hypoxia). (2) PtO2 dependent erythropoietin (EPO) production. (3) Dose‐response of EPO on erythropoietic amplification (up to two to four additional mitoses). (4) The changes of the marrow transit time.

Model simulations are compared with experimental data for changes of erythropoiesis during hypoxia, EPO‐injection, and different forms of anaemia. A satisfactory agreement suggests that the model adequately describes and correlates different direct and indirect ways to stimulate erythropoiesis. It quantifies the role and relative contribution of the haematocrit, haemoglobin concentration, atmospheric oxygen pressure, tissue oxygen pressure, and plasma volume as triggers in erythropoietic stimulation under various conditions. Furthermore, the model may allow to optimize the scheme of EPO‐administration and to find the maximum increase of erythropoiesis for a given amount of erythropoietin.
A comparison of stem cell assays using early or late spleen colonies
Authors: Nečas E. , Znojil V.
Published: May 2008

Abstract The distribution of spleen colony diameters was determined 5.5, 8.0, 10.5 and 13.0 days after injection of normal bone marrow cells to lethally irradiated recipients. A relative lack of small colonies on day 8.0, as compared with days 5.5, 10.5 and 13.0, argued against a time continuum in colony appearance. The spleen colonies observed after 10 days or more probably represented a mixture of colonies which developed from the originally transplanted CFU‐S and those arising from secondary CFU‐S. Thus, late appearing spleen colonies may not necessarily identify a different, less mature, population of CFU‐S. Administration of increasing amounts of bone marrow cells was used for comparing the linearity of the CFU‐S assay for colonies observed after 8 days or after 12 to 13 days. The influence of overlapping colonies on the results was considerably augmented if large spleen colonies were observed after 12 or 13 days. Subsequently the CFU‐S assay lost much of its quantitative character. We believe that some previously published data might have been misinterpreted by neglecting the important differences between ‘early’and ‘late’CFU‐S assays.
The cell kinetics of the epidermis and follicular epithelium of the rat: variations with age and body site
Authors: Morris G. M. , Hamlet R. , Hopewell J. W.
Published: May 2008

AbstractThe skin from rats of differing age was used to quantify variations in the cell kinetics of the epidermis and the follicular epithelium of different body sites. Four parameters were assessed, namely the basal cell density (BCD), the labelling index (LI), the duration of DNA synthesis (ts) and the basal cell turnover time (tT). The BCDs of the epidermis of the dorsum and the upper surface of the foot were similar in rats of 7, 14 and 52 weeks of age, but there was an indication of a progressive decline with increasing age in the BCD of the epidermis of the ear and tail. There were no age‐related changes in the length oftsin any of the four body regions. The rate of cell proliferation, as indicated by the values of the LI andtT, was relatively rapid in the epidermis of the dorsum, foot and tail of rats aged 7 weeks (LI > 12%;tT< 80 h). In rats aged 14 weeks this rate of proliferation was maintaned in the epidermis of the dorsum. However, in the foot and tail the rate of cell proliferation was decreased (LI < 10%;tT> 85 h). A fall in the rate of proliferation of the epidermis of the dorsum was only seen in 52‐week‐old animals. In these animals the rates of proliferation in the foot and tail were similar to those at the age of 14 weeks. In the epidermis of the ear there was no appreciable change in the rate of cell proliferation with age.

The values of the cell kinetic parameters varied in the different body sites. For example, in 52‐week‐old animals values fortTwere relatively short in the epidermis of the tail and foot and appreciably longer in the epidermis of the dorsum and ear.

Considered overall, values for the cell kinetic parameters of the epidermis were comparable with those for the follicular epithelium. The only major differences between the epidermis and the follicular epithelium were in the upper surface of the foot at 7 weeks of age, and in the tail at 7 and 14 weeks of age, where the LI was higher and thetTshorter in the epidermis than in the follicular epithelium.

The relevance of the observed age‐ and body‐site‐related variations in the cell kinetics of the epidermis are discussed in relation to previously described differential changes in the radiosensitivity of the skin in this strain of rat.
Long term growth and maintenance of stratified rat urothelium in vitro
Authors: Chlapowski F. J.
Published: May 2008

Abstract Culture conditions that allow long term growth and maintenance of rat urothelium have been determined using short (3 to 8 days) and long (14 to 60 days) term measurements of cell density and tritiated thymidine incorporation as indices. The basal nutrient medium utilized was a mixture of 199 plus Ham's F 12 (1:1) supplemented with insulin (1 μ/ml) and hydrocortisone (1 μ/ml). Long term culture of urothelium seems to require porous collagen. Porous albumin, or plastic dishes thinly coated with albumin, collagen, fibronectin or mixtures thereof, did not support long term maintenance. Serum was required at a concentration of 5%, independent of other additives. Decreasing Ca++ levels below that normally found the basal medium (∼1 × 10−3) to as low as 1 × 10−4, resulted in increased short term proliferation, but decreased long term maintenance by causing a loss of stratification of the urothelium. Even a slight increase in Ca++ concentration from 1.0 to 1.5 × 10−3 resulted in an inhibition of proliferation and an increase in the number of large flat cells which subsequently sloughed off in sheets. The deletion of either insulin, hydrocortisone or both, inhibited growth. The addition of epidermal growth factor (EGF) or its homologue, transforming growth factor (TGF‐α), increased cell proliferation markedly and caused a variable increase in stratification. However, epithelium induced to rapid growth and proliferation with EGF, eventually exhausted its growth potential and died. TGF‐β1, alone or in combination with either EGF or α‐TGF, had no additional effect upon urothelial growth. Repeated transfers of urothelium by enzymatic dissociation led to decreased growth and maintenance potential. The data indicates that long term maintenance of stratified urothelium in culture requires a porous collagen substrate and fetal bovine serum together with hormonal requirements and concentrations of Ca++ that neither greatly stimulate nor inhibit growth.
Pemphigoid, pemphigus and desmoplakin as antigenic markers of differentiation in normal and tumorigenic mouse keratinocyte lines
Authors: Kulesz‐Martin M. , Kozlowski P. , Glurich I. , Lisafeld B. , Hemedinger E. , Kumar V.
Published: May 2008

Abstract The expression of differentiation stages in a murine epidermal cell transformation model has been investigated as a basis for studies of chemically‐induced differentiation. Antibodies in sera of patients with the autoimmune diseases bullous pemphigoid and pemphigus vulgaris exhibit specific reactivity to antigenic determinants of basal and spinous cells, respectively, in sections of mouse and human epidermis. In addition, spinous cells in epidermis are reactive with a mouse monoclonal antibody to desmoplakin, a desmosomal component immunologically distinct from pemphigus. These antibodies were used to identify and attempt to quantify keratinocyte subpopulations in culture based on differentiation stage. Epidermal cell lines were cultured under conditions which favour proliferation (0.02 to 0.04 mm extracellular Ca2+, i.e. low Ca2+ conditions) or differentiation (0.1 mM to 1.4 mM Ca2+), as previously shown using primary cultures of mouse keratinocytes. Two independently‐derived normal keratinocyte lines demonstrated Ca2+‐dependent reactivity with pemphigoid and pemphigus antiserum, like that which has been observed in primary cultures. Furthermore, a Ca2+ and time‐dependent reactivity with the three antisera was also observed in a papilloma cell line (derived from one of the normal cell lines after treatment in vitro with 7,12‐dimethylbenz[α]anthracene). Papilloma cells cultured under conditions of low extracellular Ca2+ were comprised of three subpopulations: cells reactive only with pemphigoid anti‐serum, cells reactive with pemphigoid and desmoplakin antibody (intracellular location), and cells reactive only with desmoplakin antibody. However, like the normal cell lines, papilloma cells underwent a transition to predominantly a spinous cell population (i.e. reactive with pemphigus and desmoplakin antibody) in response to extracellular Ca2+. A slower loss of pemphigoid antibody reactivity was noted in papilloma cells, consistent with an abnormal regulation of differentiation. The attempt to characterize these dynamic transitions from basal to spinous cell subpopulations in culture was considered to be prerequisite for the use of the model to investigate differentiation‐inducing agents in carcinoma therapy.
The measurement of villus cell population size in the mouse small intestine in normal and abnormal states: a comparison of absolute measurements with morphometric estimators in sectioned immersion‐fixed material
Authors: Wright N. A. , Carter J. , Irwin M.
Published: May 2008

Abstract From a functional viewpoint, the most important part of the small intestinal mucosa is the villus epithelium; in the experimental study of intestinal adaptation it is often necessary to estimate the size of the population. For these reasons, a systematic study of methods of measuring the size of the villus cell population was undertaken in villi of normal morphology from control animals, and in villi of abnormal morphology as produced by treatment of mice with cytosine arabinoside (Ara‐C).

The villus cell population can be measured with precision and accuracy in both normal and abnormal mucosal states, with a relative standard error usually less than 5%, with good reproducibility, and with very low counting errors. In the mouse a practically linear relationship between the villus cell population size and position in the small intestine can be shown. In normal, control animals, linear estimates of villus population, for example the villus height, the villus core height, and the villus row count measured in sections of immersion‐fixed material, were found to be rough approximations as indicators of the villus cell population, and were highly correlated with it. Product variables, comprising the products of a height with a width measurement, show a large improvement over the linear variables as estimators of villus cell population. In populations of abnormally shaped villi produced by Ara‐C, both linear and product variables showed a considerable decrease in correlation with the villus cell population. Consequently, indirect, linear measurements of the villus population size do not accurately reflect the size of the villus population in abnormal villi.

Finally, a method comparing theoretical and measured surface: volume indices was used to indicate that the most appropriate shape for normal mouse villi is a simple cylinder; this method would also be applicable for other villus cell populations.
Probability of self‐renewing divisions of spermatogonial stem cells in colonies, formed after fission neutron irradiation
Authors: van Beek M. E. A. B. , Meistrich M. L. , de Rooij D. G.
Published: May 2008

Abstract.

Repopulating spermatogenic colonies, found in the seminiferous epithelium after irradiation with fast‐fission neutrons, were studied to determine the cha nce that a stem cellAsingle (As spermatogonium would complete a self‐renewing division (P). Mathematical formulas originally derived for such studies in haemopoietic colonies were employed, and a method specifically aimed at spermatogenic colonies was developed. The results showed that during the first division after irradiation, P is close to 1 ‐0. P decreases in later generations, but remains 0‐7 or higher up to the 4th or 5th divisions. The mean value for P was over 0‐8, which is higher than the value of 0‐6‐0‐7 found for stem cells in haemopoietic colonies.
A morphometric analysis of individual cell death in bronchial carcinoids
Authors: Al‐Saffar N. , Moore J. V. , Hasleton P. S.
Published: May 2008

Abstract. The incidence and morphometric characteristics of individual dead cells have been measured in 51 cases of broncho‐pulmonary carcinoid tumours. In both typical and atypical carcinoids, these dead cells were distinguished by nuclei that were significantly smaller and less regular than those of ‘intact’ tumour parenchymal cells. The proportion of dead to all tumour cells was not significantly different for typical and atypical carcinoids (17 and 13%, respectively). For 33 of these tumours, their ploidy status had also been established. In diploid tumours, the proportion of dead cells was 18% and in aneuploid tumours 12%. The prognosis of patients with atypical carcinoids was significantly worse and such tumours were more commonly aneuploid. Thus the incidence of individual cell death does not appear to be positively associated with poor prognosis in this series. The association between ‘necrosis’ and poor prognosis commented on in the literature may relate more to a different form of cell death, expressed histopathologically as gross coagulative necrosis, the incidence of which is significantly higher among the atypical, aneuploid tumours.
Ultraviolet B irradiation induces epidermal regeneration with rapidly cycling cells
Authors: Olsen W. M.
Published: May 2008

Abstract. The left flank of hairless mouse skin was irradiated with a minimal erythema dose of ultraviolet B (UVB) light at 297 nm (25 mJcm‐2), while the right flank served as untreated control. The alterations in epidermal growth kinetics induced by this UVB dose were studied with the percentage of labelled mitoses (PLM) technique during the period of increased proliferation. Thirty hours after irradiation, when a large cohort of cells appears in S phase, each animal was injected intra‐peritoneally with 50 /iCi tritiated thymidine ([3H]‐TdR). The number of labelled basal and suprabasal cells, as well as their localization in epidermis were registered in histological sections at short intervals up to 48 h after the [3H]‐TdR pulse. Labelled mitoses were also counted in the same specimens. The results showed a four‐fold increase of the high initial number of labelled cells in UVB‐exposed epidermis within 18 h of the pulse injection, and a sixfold increase after 36 h. In control epidermis, where the starting value of the labelling index was much lower, there was only a three to four‐fold increase in the number of labelled cells during the period studied. The PLM and the labelling index data were consistent with an average cell cycle time of approximately 10–12 h for UVB‐exposed cells, in contrast to about 30 h for the fastest cycling population in control epidermis. The PLM curve also indicated a prolonged S phase duration in UVB‐exposed epidermis compared with controls. In addition, labelled cells were seen in the suprabasal layer as early as 6 h after the [3H]‐TdR injection and within 36 h labelled cells had reached the outermost layer of nucleated cells, indicating a reduced transit time through epidermis. The present study shows that a minimal erythema dose of UVB light at 297 nm induced a period of increased transit time through the S phase, combined with rapid cell proliferation, leading to an overall shortening of the epidermal cell cycle time. The cohort of cells labelled with [3H]‐TdR 30 h after irradiation seemed to proceed as a wave of partially synchronized cells through the cell cycle for more than two rounds, which is comparable with the cell kinetic perturbations observed in regenerating mouse epidermis.
Vasopressin‐enhanced urea transport by rat inner medullary collecting duct cells in culture
Authors: Schwartz J. H. , Bengele H. H. , McNamara E. R. , Alexander E. A.
Published: May 2008

Abstract. The distal inner medullary collecting duct (IMCD) is critical in the urinary concentrating process, in part because it is the site of vasopressin (AVP)‐regulated permeability to urea. The purpose of these experiments was to develop a cell culture model of the IMCD on permeable structure and to characterize the responsiveness to AVP. Rat IMCD cells were grown to confluence on collagen‐coated Millipore filters glued onto plastic rings. To assess the time required to achieve confluence, the transepithelial resistance was measured periodically and was found to be stable after 2 weeks, at a maximal value of 595 ± 22 ω cm2. In separate monolayers the effect of AVP on inulin and urea permeability was determined. While inulin permeability was unchanged after AVP, urea permeability increased from 6.0 ± 0–4 to peak values of 16.0 ± 3–8(10nM),23.1 ± 3–9(1 μM)and28 1 ± 4–9(10μM) X 10‐6cms‐1 (n= 24). In 10 other monolayers, after the addition of 1 mM 8‐Br‐cAMP, urea permeability increased from 5.1 ±0–3 to 8.1 ± 1–6 times 10‐6 cm s‐1 and, after 8‐Br‐cAMP +3‐isobutyl‐l‐methylxanthine, to 12.2 ± 0–7 times 10‐6 cms‐1. We conclude that rat IMCD cells grown in culture exhibit the characteristics of a ‘tight’ epithelium. Inulin and urea permeability are not different in the absence of AVP, consistent with high resistance junctional complexes. Furthermore, IMCD cells retain the capacity for AVP‐regulated urea permeability, a characteristic feature of this nephron segment in vivo.
Specific expression of proteins and phosphoproteins in 3T3 T mesenchymal stem cells at distinct growth arrest and differentiation states
Authors: Sparks R. L. , Zschunke M. A. , Seibel‐Ross E. I. , Tracy R. , Zalitis J. G. , Boman B. M. , Hoerl B. J. , Scott R. E.
Published: May 2008

Abstract. Murine mesenchymal stem cells can be induced to arrest their growth at a series of growth and differentiation states in the G1 phase of the cell cycle. These include the predifferentiation arrest state (GD) at which the integrated control of proliferation and differentiation is mediated, the growth factor/serum deficiency arrest state (GS), and the nutrient deficiency arrest state (GN). Cells at states of reversible nonterminal differentiation (GD́) and irreversible terminal differentiation (TD) can also be isolated. In this paper we have employed 1‐ and 2‐dimensional (D) gel electrophoresis to evaluate changes in specific proteins that occur during the various growth and differentiation states of 3T3 T mesenchymal stem cells. The protein composition of membrane, microsome and cytosol preparations of cells arrested at GD, GS and GN states was determined by 2‐D gel electrophoresis. More than 50 distinct polypeptides could be identified for each arrest state in gels analysed by a silver staining procedure or by autoradiography following [35S]‐methionine labelling. A second series of studies established that a more limited number of differences could be identified if phosphoproteins were analysed by 1‐D gel electrophoresis in cells at the GS, GD, GD́. and TD states. These results established that one distinct 37 kD phosphoprotein is present in all growth arrested cells and that two distinct differentiation‐associated phosphoproteins with molecular weights of 29 kD and 72 kD are present in cells at the GD́ and TD states. Thus, the composition of proteins and phosphoproteins in mesenchymal stem cells serves to characterize different states of growth arrest and differentiation. The identification of differential protein expression provides an opportunity to test their functional role in growth and differentiation control.
In vivo effect of the tetrapeptide, N‐Acetyl‐Ser‐Asp‐Lys‐Pro, on the G1‐S transition of rat hepatocytes
Authors: Lombard M.‐N. , Sotty D. , Wdzieczak‐Bakala J. , Lenfant M.
Published: May 2008

Abstract. The synthetic molecule N‐Acetyl‐Ser‐Asp‐Lys‐Pro (Ac‐SDKP), corresponding to the low molecular weight inhibitory factor preventing in vivo haematopoietic stem cell (CFU‐S) entry into DNA synthesis, was tested in two heterologous systems in vivo: adult regenerating rat liver and 10‐day‐old rat hepatocytes synchronized by an irritating trigger. In both systems, it was shown that doses of 2–8 μg kg‐1 of tetrapeptide inhibited 50–70% of the hepatocyte G1‐S transitions.
Haemopoietic stem cells: spleen colony‐forming cells are normally actively proliferating
Authors: Nečas E. , Znojil V. , Šefc L.
Published: May 2008

Abstract. The haemopoietic stem cells forming spleen colonies (CFU‐S) had on average 30 to 40% of cells engaged in the DNA synthesis in normal mice continuously over 4 years. A majority of experiments aimed at the suppression of the CFU‐S proliferation, which included suppression of the T‐lymphocytes by means of cyclosporin A or by adult thymectomy, administration of antibacterial and antifungal agents and maintainance of mice in a sterile environment, suppression of antibody‐producing cells by a successive administration of the bacterial lipopolysaccharide and cyclophosphamide and attempts to increase the total number of CFU‐S in the body through massive transfusions of bone marrow cells or by grafting plugs of the bone marrow under the kidney capsulae, have not been sufficiently effective. A transient suppression of CFU‐S proliferation occurred during recovery of the haemopoietic tissue from damage caused by cyclophosphamide. The results support the view that changes in CFU‐S numbers and in the proportion of them in DNA synthesis may be positively correlated when CFU‐S numbers fluctuate physiologically about their normal values. The failure to manipulate the CFU‐S proliferation rate easily suggests that proliferation of these cells may not be under a strong ‘switch on ‐ switch off’ control.
Corrections in counting paired nuclei labelled with bromodeoxyuridine in tissue sections
Authors: Wheeler J. , Matthews J. N. S. , Morley A. R.
Published: May 2008

Abstract. Bromodeoxyuridine is finding increasing use as an alternative to, or in conjunction with, tritiated thymidine for labelling nuclei in DNA synthesis. Precise identification of labelled nuclei is possible, even when there is considerable overlap between neighbouring nuclei. In the sparsely labelled renal cortex of the normal male mouse, ‘flash labelling’ with bromodeoxyuridine shows single labelled nuclei at 1h. At 24, 48 and 72 h after injection of bromodeoxyuridine, some labelled cells are seen to lie adjacent and such labelled pairs are presumed to be the result of cell division. Single labelled nuclei at 24, 48 and 72 h might indicate arrest in DNA synthesis or a prolonged G2 period, but it is important to recognize that a correction must be made for paired labelled nuclei in which one member is out of the plane of section. The factors involved in making such a correction are discussed and a correction table calculated. In the normal male mouse renal cortex, we show that nearly all cells labelled at 1 h had divided by 72 h.
Differentiation‐related regulation of 1,25 dihydroxy vitamin D3 receptor mRNA in human leukaemia cells HL‐60
Authors: Pan P. , Reddy K. , Lee S. , Studzinski G. P.
Published: May 2008

Abstract. Vitamin D receptor (VDR) is a nuclear protein which mediates the physiological actions of its hormone ligand, 1, 25‐dihydroxyvitamin D3 (1, 25(OH)2D3). While it appears that the receptor‐hormone complex regulates the expression of hormone‐dependent genes involved in mineral homeostasis, its role in induction of differentiation of leukaemic cells is less clear. We have studied the expression of the VDR gene in several sublines of HL‐60 leukaemic cells with varying responsiveness to 1, 25(OH)2D3. Sublines which rapidly differentiated to monocytic forms were shown to contain elevated steady‐state levels of VDR mRNA within 1 h of exposure to high concentration of 1, 25(OH)2 D3. This up‐regulation of the expression of VDR was not apparent in sublines in which monocytic differentiation occurred after a delay of several days. Beginning at approximately 3 h after exposure to 1, 25(OH)2 D3 in most cases, there was a gradual decline in VDR mRNA levels. Measurement of steady‐state levels of mRNA for c‐myc and c‐fos showed that in sublines of HL‐60 cells which respond rapidly to 1, 25(OH)2D3, elevation of VDR mRNA is evident prior to the changes in proto‐oncogene expression. These data are consistent with the hypothesis that a change in VDR gene expression is one of the steps that promote monocytic differentiation.
Cell proliferation in human tumour xenografts: measurement using antibody labelling against bromodeoxyuridine and Ki‐67
Authors: Parkins C. S. , Bush C. , Price P. , Steel G. G.
Published: May 2008

Abstract. Cell proliferation was investigated in human tumour xenografts using bromodeoxyuridine (BrdUrd) labelling, evaluated either by flow cytometry or in tissue sections, and also using the proliferation marker Ki‐67. BrdUrd labelling was found to increase when cryostat tumour sections were digested with an enzymic solution. This yielded a labelling index up to four times higher than that obtained using the flow cytorneter. Ki‐67 indices were found to be higher than those reported for human tumour biopsies, as may be expected due to the enhanced growth rate of the xenografts. Significant heterogeneity was observed in the results for cervix, breast and bladder tumours, and the results of the three methods were poorly correlated. However, three of the four tumour types showed that the tumour with the lowest Ki‐67 index also had the longest potential doubling time. Since the measurement of Ki‐67 index was found technically easier to perform, and also adequately reflects relative tumour cell proliferation, it is preferred over the other techniques.
In vivo regulation of granulocyte protluction in acute inflammation
Authors: Hamood M. , Fondu P.
Published: May 2008

Abstract. In order to study in vivo the enhanced granulopoiesis that occurs during acute inflammation, 1–3 sterile metallic copper rods were inserted subcutaneously into mice either at the same place (one abscess) or at different sites (multiple abscesses). Diffusion chambers filled with bone marrow cells were implanted intraperitoneally for 3 days. When a single abscess was created, the granulocytic content of the diffusion chamber increased similarly whatever the number of inserted copper rods. However, there was a direct relationship between the number of abscesses and the number of granulocytic cells harvested from the diffusion chambers.

In order to investigate the role of T‐lymphocytes in the production of diffusible stimulating factors that act on diffusion chamber granulopoiesis, cyclosporin A (CyA) was given to the mice with implanted copper rods. CyA abrogated the induced enhancement of CFU‐S, CFU‐GM and mature granulocyte numbers inside the diffusion chamber. The stimulatory effect of inflammation on diffusion chamber granulopoiesis was not observed in T‐lymphocyte‐deficient nude mice.

These data suggest that in vivo stimulation of granulopoiesis is related to the level of inflammation, and that this effect requires the functional integrity of T‐lymphocytes.
C‐myc expression is maintained during the G1 phase cell cycle block produced by beryllium
Authors: Skilleter D. N. , Barrass N. C. , Price R. J.
Published: May 2008

Abstract. Salts of the toxic metal beryllium have been shown previously to prevent the synthesis of several enzymes essential for DN A replication in proliferating rat hepatic cells in vivo, and to inhibit the division of rat liver‐derived BL9L epithelial cells in vitro, specifically during the G1 phase of the cell cycle. The present study shows, however, that exposure of serum‐stimulated sub‐confluent monolayer cultures of synchronized BL9L cells to inhibitory concentrations of the beryllium salt BeSO4 (50 μm) did not impair expression of the cell proliferation associated nuclear proto‐oncogene c‐myc. On the contrary, the increased c‐myc mRNA levels normally observed during the G1 phase were maintained by continuous exposure of the cells to BeSO4. This response was specific in that other colloid forming metal salts (ZnSO4 and ZrSO4), which did not inhibit cell division, had no affect on c‐myc expression, and mRNA levels for the constantly expressed H‐2Kb major histocompatibility complex gene (3'Kb) were unaltered by BeSO4 treatment of the cells. The prevention by Be2+ of the down‐regulation of c‐myc expression in serum‐stimulated BL9L cells appears to result from a modulation of the endogenous transcriptional control process for c‐myc, which allows a maintained expression of the gene.
Inheritance and regression toward the mean in heterogeneous cell populations
Authors: Gamel J. W. , Axelrod D. E.
Published: May 2008

Abstract. Traits such as birth size and lifetime can vary widely even among non‐mutated progeny of the same cell proliferating in the same environment. On the other hand, population parameters of these traits may remain stable over many generations, and there may be a distinct inheritance of these traits from mother to daughters. We have reconsidered the implication of mother‐daughter correlations in light of linear regression analysis. It is proposed that a non‐mutant cell whose phenotype deviates from the population mean produces progeny whose rate of regression toward the mean is proportional to 1‐r, where r is the mother‐daughter correlation coefficient of the trait under study. Theoretical support for this proposition is derived from linear regression analysis. Empirical support is found in pedigree analysis of cell growth constants among NIH3T3 mouse fibroblast cells, where the presence of an activated human ras oncogene is associated with a decreased r and an increased rate at which the growth constants of progeny regress toward the population mean.
Importance of cell cycle perturbations on the effectiveness of N‐methylformamide and anti‐neoplastic drugs in combination
Authors: Cucco C. , D'Agnano I. , Marangolo M. , Candiloro A. , Zupi G.
Published: May 2008

Abstract. The effect of N‐methylformamide (NMF) in combination with Adriamycin (ADM) and cis‐diamminedichloroplatinum (DDP) on the cell survival and cell cycle kinetics of two human tumour lines was assessed: HT29 colon carcinoma and M14 melanoma cells were exposed to ADM and DDP alone or in combination with a non‐cytotoxic dose of NMF, according to different schedules.

The results demonstrate that NMF exposure sensitized both tumour cell lines to the lethal activity of ADM and DDP; however, reverse sequences had to be applied to reach an increase in the lethal activity of the two different drugs.

The ADM‐NMF combination determined a powerful decrease in the surviving fraction of the two cell lines when ADM was given as the first agent (ADM → NMF), while the reverse sequence did not increase the ADM cytotoxic effect. With respect to the DDP‐NMF association, the sequence which accounted for a greater sensitizing effect was NMF administration followed by DDP treatment (NMF → DDP).

This work demonstrates the importance of timing in combined treatments which involve NMF. A delay in cell proliferation elicited by NMF exposure could be responsible for the effectiveness of the combined treatment.
Caffeine reverses the cytotoxic and cell kinetic effects of Novantrone (mitoxantrone)
Authors: Traganos F. , Kaminska‐Eddy B. , Darzynkiewicz Z.
Published: May 2008

Abstract. Caffeine is known to potentiate the cytotoxicity of a variety of DNA damaging agents presumably by reducing the ability of the cells to repair potentially lethal lesions. However, in the present study we observe that 5 mM caffeine reverses the cell kinetic and cytotoxic effects of the intercalating drug Novantrone (mitoxantrone) on L1210, HL‐60 and CHO cells. Novantrone alone, at a concentration of 20–30 ng/ml, given to cultures for 1 h, inhibits cell growth by about 50% and causes cells to accumulate in S and G2 phase and to enter a higher DNA ploidy level. Treatment of these cell lines with 5 mM caffeine alone for 1 h has a minimal effect on cell proliferation; suppression of cell growth varies from 5 to 10%. Exposure of cells to Novantrone for 1 h in the presence of caffeine results in a significant reduction of the Novantrone effects; the cell growth rate is partially restored (e.g. caffeine reduces suppression of L1210 cell growth from 48 to 83% of control) and in each of the cell lines studied, the Novantrone‐induced cell accumulation in S and G2 is abolished. Combined treatment with caffeine and Novantrone also increases the clono‐genicity of CHO cells 8·5 times over that seen in cultures treated with Novantrone alone. In contrast to the combined treatment with caffeine + Novantrone, pretreatment of cells with caffeine provides no protection. Likewise, post‐treatment with caffeine provides little reversal of growth inhibition and G2 cell accumulation, especially if the post‐treatment is delayed in time. The present data, in conjunction with evidence in the literature that caffeine protects cells against the cytotoxic effects of doxorubicin, suggest that caffeine may play a more general role in protecting cells against planar aromatic molecules such as intercalating agents.
Protein expression in relation to the cell cycle of exponentially growing human prostatic epithelial cells
Authors: Herz F. , Czerniak B. , Deitch D. , Wersto R. P. , Simmons D. A. , Koss L. G.
Published: May 2008

Abstract. This report concerns the study of the relationship between protein expression and the cell cycle in exponentially proliferating benign and malignant human prostate epithelial cells in short‐term cultures. Multiparameter flow cytometric measurements were performed to correlate the expression of prostate‐specific acid phosphatase, epithelial membrane antigen and epitectin with cell cycle progression. The expression of the three proteins was heterogeneous in G1 cells. The early post‐mitotic cells exhibited the lowest levels when compared with late G1 cells, wherein the expression was many times greater. There was no further increase as the cells progressed through S and G2+ M. These findings, corroborating prior observations in other systems, suggest the possibility that the levels of the proteins studied increase during the G1 phase of the cell cycle and drop during or immediately after cytokinesis. As an alternate explanation, the heterogeneity of protein expression characteristic of G1 cells may be due, at least in part, to an asymmetric apportionment of cell constituents at mitosis.
Growth and cell kinetics of human hair papilla cells in vitro. An autoradiographic and flow cytometric study
Authors: Bassukas I. D. , Arai A. , Schell H. , Hornstein O. P.
Published: May 2008

Abstract. Hair papilla, a distinct specialized dermal compartment, plays a fundamental role in the biology of hair growth. Recently some attention has been focused on hair papilla cells (HPC) as possible targets for drugs influencing the hair growth. Isolation and cultivation of the HPC facilitates screening for such drugs. In the present work, growth and cell kinetics of human occipital scalp follicle HPC have been studied in vitro. HPC grow according to a Gompertz function, i.e. with considerable growth delay long before becoming confluent cultures, due probably to elongation of the potential doubling time (Tpot) and to a parallel increasing cell loss rate. The [3H]dT labelling index of the HPC strongly depends on the age of the subculture; the cycle time being about 4 days. A potential doubling time of about 93 h, indicative of growth fraction (GF) =1, and a duration of S phase and G2+ M phase of about 8 h each were found by the combined application of continuous labelling with [3H]dT and DNA flow cytometry.
Plasma membrane and intracellular pools of transferrin receptors decline during in vitro cultivation of U937 cells
Authors: Salcedo T. W. , Fleit H. B.
Published: May 2008

Abstract. Transferrin receptor expression in the monocyte‐like cell line U937 was investigated during in vitro cultivation. U937 cells expressed a single class of high affinity surface transferrin receptors (Kd≅ 4nm), with apparent subunit M, of 90–95 000 Da as determined by SDS‐reducing PAGE. [125I]‐transferrin binding studies on detergent‐solubilized cells revealed that half to two‐thirds of the total functional binding sites were located intracellularly. Radioligand binding, immuno‐fluorescence and flow cytometry studies were performed on intact, detergent‐solubilized, or saponin‐permeabilized cells, using either transferrin or the anti‐transferrin receptor monoclonal antibody OKT9 IgG. These studies demonstrated that functional and antigenic transferrin receptor levels were maximal on cells 24 h after subculture at low density and declined during the culture period. Scatchard analysis of radioligand binding data suggested that the decline in functional transferrin binding sites resulted from a decline in the number of available receptors. These results demonstrate that in U937 cells there is a density‐dependent regulation of transferrin receptor expression, resulting in a loss of functional and antigenic receptors from both plasma membrane and intracellular locations.
Feasibility and reproducibility of the [3H]‐thymidine labelling index in breast cancer
Authors: Silvestrini R.
Published: May 2008

Abstract. For several malignancies, autoradiographic evaluation of the S phase cell fraction by means of [3H]‐thymidine ([3H]dT) has proved a valid prognostic tool which should be included in a ‘risk factor profile system’ to estimate prognosis in individual patients. However, its clinical implementation requires a methodological standardization, a high feasibility and an assessment of the reproducibility of results, within and between different laboratories. The recent availability of a kit for in vitro incubation with [3H]dT and histological fixation of solid tumour specimens has contributed to the methodological simplification and standardization of the technical procedure, increasing its feasibility in several institutions. For breast cancer, preliminary results from a quality control study promoted by the Italian Society of Basic and Applied Cell Kinetics (SICCAB) showed a high inter‐observer reproducibility within (r = 0·96) and between different laboratories (r = 0·93). Moreover, on a series of 20 autoradiographic samples sent to 15 different institutions, only 2% of tumours shifted from one of the cell kinetic subgroups, defined using the median [3H]dT labelling index (LI) of 2·8% as a cutoff, to the other. In addition, a marked consistency of results was observed among the different Italian institutions involved in cell kinetic studies on breast cancers, both in terms of median [3H]dT LI values and basic correlations. Periodic assessment of the reproducibility of [3H]dT LI evaluation among different laboratories is needed, especially for institutions involved in multi‐centre clinical protocols based on cell kinetic characteristics.
Expression of cell cycle related proteins—proliferating cell nuclear antigen (PCNA) and statin—during adaptation and de‐adaptation of EUE cells to a hypertonic medium
Authors: Pellicciari C. , Danova M. , Giordano M. , Conti A. M. Fuhrman , Mazzini G. , Wang E. , Ronchetti E. , Riccardi A. , Romanini M. G. Manfredi
Published: May 2008

Abstract. EUE cells adapted to grow for long times in a hypertonic medium have a longer cell cycle than those growing in isotonic medium. To elucidate whether this lengthening involves specific cycle phases to differing extents, the expression of two cycle‐related protein, PCNA and statin, was studied by dual parameter flow cytometry of indirect immunofluorescence protein labelling and DNA content. In isotonic medium, most cells, in all the cycle phases, were PCNA positive; in contrast, PCNA negative cells and statin positive cells were very few in number and only fell in the G0/l range of DNA contents. In hypertonic medium, the frequency of PCNA positive cells was lower, and that of statin positive cells higher, than in isotonic medium, particularly in the Go/1 range of DNA contents: this suggests that a G0 block occurs under long‐term hypertonic stress. Consistently, dual parameter flow cytometric measurement of BrdUrd immunofluorescence labelling and DNA content showed that fewer cells entered S phase in hypertonic medium and their progression through the S phase was slower; evidence was also found for the occurrence of a G2 block. These kinetics changes were fully reversible in isotonic medium, thus indicating the adaptive nature of the EUE response to hypertonicity.
Automatic classification of cells in cell cycle phases based on Ki‐67 antigen quantification by fluorescence microscopy
Authors: Guillaud P. , Vermont J. , Seigneurin D.
Published: May 2008

Abstract. Ki‐67 antigen is thought to be a marker of cell proliferation, as it can be detected in cycling cells, i.e. cells in G1, S, G2 and M phases, but not in resting cells. The immunocytochemical staining pattern obtained by the Ki‐67 monoclonal antibody varies, depending on the cell cycle phases. Analysis of double staining of Ki‐67 antigen and DNA in the MCF‐7 cell line by videomicrofluorometry allows the description of both the level and the pattern of Ki‐67 staining in the form of a set of parameters defining each cell. These parameters were measured in MCF‐7 cell populations characterized according to their position in the cell cycle. They were submitted to a statistical analysis (principal component and discriminant analysis) which allowed the determination of the optimal parameters to characterize a given cellular group and permitted the use of these parameters for an automatic classification of cells in the different cell cycle phases. In G1, S, G2, prophase + metaphase and anaphase + telophase cells, these parameters allowed a classification of cells with a good‐classification rate of 94·37%. A comparison of this method with methods based on the DNA histogram and bromodeoxyuridine uptake was performed. The classification coefficients stemming from the discriminant analysis were introduced into a program to obtain, automatically, the Ki‐67 labelling index and the percentages of cells in each phase. This method, which allows a quick evaluation of the proliferation and the phase indices, may be more widely applicable.
Stimulator of proliferation of spleen colony‐forming cells in T‐cell deprived mice treated with cyclophosphamide or irradiation
Authors: Milenković P. , Ivanović Z. , Kataranovski M. , Lukić M. L.
Published: May 2008

Abstract. A role for T‐cells in the regulation of CFU‐S proliferation was investigated by determining the presence and activity of CFU‐S proliferation stimulator (CFU‐S stimulator) in adult mouse bone marrow after irradiation or cyclophosphamide (Cy) treatment. CBA mice previously deprived of T‐cells by thymectomy, irradiation and bone marrow reconstitution (TIR) were thereafter treated with 4·5 Gy irradiation or 200 mg/kg Cy. Regenerating bone marrow cells of TIR and corresponding control mice after irradiation or Cy treatment produced CFU‐S stimulator. The dose dependent increase in cytosine arabinoside cell death of normal bone marrow day 8 CFU‐S was found when both CFU‐S stimulators obtained after irradiation of TIR or corresponding control animals were tested. CFU‐S stimulator activity in the bone marrow of TIR‐Cy treated mice was also detected, but the effect was not dose‐dependent. This was not related to the presence of an inhibitor of CFU‐S proliferation. It appears that the CFU‐S stimulator activity is not related to IL‐6, IL‐1 or IL‐2, or to an inhibitor of IL‐6 or IL‐1 activity. The results demonstrate the existence of CFU‐S proliferation stimulator unrelated to the two major monokines in the bone marrow of immunosuppressed mice.
Measurement of cell proliferation by enzyme‐linked immunosorbent assay (ELISA) using a monoclonal antibody to bromodeoxyuridine
Authors: Perros P. , Weightman D. R.
Published: May 2008

Abstract. An ELISA was developed and optimized to measure cell proliferation using a monoclonal antibody to bromodeoxyuridine (BrdUrd).

Incorporation of BrdUrd into myoblast monolayers, measured as the optical density at 492 nm, increased in response to fetal calf serum, IGF‐I and EGF, the ELISA data correlated closely with data obtained by BrdUrd immunocytochemistry (r = 0·984), cell counting (r = 0·972) and tritiated thymidine uptake by liquid scintillation counting (r = 0·990).

The BrdUrd ELISA is a useful alternative to measurement of tritiated thymidine uptake by scintillation counting, and has the added advantages of dispensing with the use of radioactivity and of being less labour intensive.
Verhulstian analysis of the growth of transplantable mammary tumours in sialoadenectomized mice
Authors: Leith J. T. , Harrigan P. , Michelson S.
Published: May 2008

Abstract. In this report we have analysed data published in 1989 by Inui et al. (Incidence of precancerous foci of mammary glands and growth rate of transplantable mammary cancers in sialoadenectomized mice. J. Natl Cancer Inst. 81, 1660) involving the effects of perturbation of the epidermal growth factor (EGF) status of mammary tumour‐bearing mice on subsequent volumetric responses. Removal of an endogenous EGF stimulus by surgical ablation of the submaxillary glands, the major EGF‐producing organ in mice, produced significantly slower growth of rodent mammary neoplasms, decreased success rate of transplantation, and an increase in the latent period before growth occurred. Administration of i.p. EGF (5 μg/mouse/day) to sialadectomized tumour‐bearing mice would however, increase tumour growth rate. Data were analysed using the Verhulst equation which indicated that the observed effects on tumour volumetrics by either sialoadenectomy or EGF administration could be interpreted as being produced through paracrine pathways. The use of the Verhulstian analysis indicates that it is possible to analyse neoplastic responses and infer whether paracrine or autocrine pathways are involved.