bluuebunny/crossref_metadata_2025 huggingface dataset:
A method for determination of iododeoxyuridine substitution of thymidine using reversed‐phase high‐performance liquid chromatography
Authors: Yabuki H. , Nath R.
Published: May 2008

Abstract. An assay for thymidine substitution by iododeoxyuridine (IdUrd) using reversed‐phase high‐performance liquid chromatography (HPLC) has been developed. Three principal steps in this procedure are: extraction of DNA from cell or tissues, hydrolysis of DNA into deoxynucleosides and separation using HPLC. Approximately 1 μg of DNA was recovered from 105 cells by phenol extraction, and subjected to hydrolysis into deoxynucleosides which required a three‐stage DNA digestion using enzymes DNAse I, phosphodiesterase I and alkaline phosphatase. The deoxynucleosides were separated on the Microsorb C18 column with isocratic elution; 90–100% of the DNA was recovered as deoxynucleosides on the column. The method was used to determine quantitatively the percent IdUrd substitution of thymidine in Chinese hamster lung cells in vitro and BA1112 rhabdomyosarcoma in WAG/Rij rats perfused with IdUrd. It was possible to determine the thymidine substitution by IdUrd as small as 1% using a few micrograms of DNA. The close correspondence between the percent substitutions determined by HPLC and those determined by radioactive assay using [125I]‐labelled IdUrd, confirmed the accuracy of our HPLC method. The HPLC analysis is especially suitable for the determination of percent IdUrd substitution of thymidine in tissue biopsies from animals used in in vivo experiments or humans undergoing radiation treatment.
Alteration of cell cycle kinetics by reducing agents in human peripheral blood lymphocytes from adult and senescent donors
Authors: Kubbies M.
Published: May 2008

Abstract. For improving cell proliferation reducing agents are routinely used as medium supplements in murine cell cultures, however, they are rarely used for human peripheral blood lymphocytes (PBLs). Data on changes in cell kinetics induced by reducing agents are not available. Here cell kinetic alterations induced by reducing agents in human lymphocytes are revealed by applying flow cytometric BrdUrd/Hoechst cell cycle analysis and by using the exit kinetic model of Smith and Martin. Applying alpha‐thioglycerol (a‐TG) as a model compound it was shown that the major cell kinetic effect is a shortening of the mean duration of the G0/G1 phase. The minimum G0/G1 phase duration and the percentage of the non‐cycling G0/G1 cell fraction decrease only slightly. Moreover, a lower number of PBL's are arrested in the G2/M phase of the 1st cell cycle. The durations of the S and G2/M phase in the 1st and G1 phase in the 2nd cycle are not affected. These cell kinetic effects are identical for lymphocytes from both adult and senescent donors. The supplementation of the cell cultures with recombinant IL‐2 did not induce similar cell kinetic alterations compared with a‐TG. This indicates that the variation of the cell cycle progression factor IL‐2 is not solely responsible for improvement of the cell activation process in the G0/G1 phase.
The multi‐site tumour transplantation model for human endometrial carcinoma: a statistical evaluation
Authors: Heitjan D. F. , Derr J. A. , Satyaswaroop P. G.
Published: May 2008

Abstract. We studied the effect of multi‐site tumour transplantation on tumour growth by implanting varying numbers of EnCa 101 human endometrial tumours in athymic mice. One treatment group received a single tumour per mouse, another group received two tumours per mouse and a third group received four tumours per mouse. Tumour growth was sustained in all animals by implantation of oestradiol‐17β pellets. We observed positive correlation between tumours within the same mouse, which implies that individual tumours are not statistically independent. The correlation is sufficiently large that failure to account for it in statistical design and analysis could result in studies with insufficient power and in spurious assertions of significance. Regression modelling of tumour growth curves showed that mean tumour volume per animal is not affected by the number of tumours growing on the animal; that is, the data are consistent with the null hypothesis that mean tumour volume is the same regardless of the number of tumours present. Our results therefore suggest that the use of multiple tumours per animal can increase the precision of experiments without loss of validity and at relatively little cost. However, correct and efficient analysis of the data so obtained requires more sophisticated techniques than those—such as fixed‐effects analysis of variance and the two sample t‐test—that assume independence of tumours.
Kinetics of the calcium induced stratification of human keratinocytes in vitro
Authors: Heenen M. , Graef Ch. De , Galand P
Published: May 2008

Abstract. In a low concentration of calcium (0.1 mM), keratinocytes form a monolayer with about 30% of cells synthesizing involucrin. After addition of calcium to the culture medium to a concentration of 1.2 mM, the monolayer stratifies within 24 h, with a preferential migration of involucrin positive keratinocytes. In the present study, we tried to determine if keratinocytes control the decision to migrate at a distinct cell cycle point. A percentage labelled mitosis (PLM) curve was constructed for keratinocytes grown in low calcium medium and values for the length of the cell cycle (47 h), S phase duration (11 h) and G2+ M period (6 h), were obtained. Monolayer cultures at 80% confluence were switched to high calcium concentration at various times (from 0 to 48 h), after pulse labelling with [3H]‐thymidine. Based on the PLM data, the behaviour of cells known to be in S, G1 and G2 at the time of the migration stimulus were followed. No significant difference in the percentage of labelled suprabasal cells was found for any point of the cell cycle. For cells submitting to stratification, in S phase involucrin staining showed that about 60% of the [3H]‐thymidine labelled cells were also involucrin negative. These results indicate that upward migration of keratinocytes in cultured epithelium can be triggered at all points in the cell cycle with equal probability and is not restricted to those cells that already contained involucrin.
Mathematical modelling of microenvironment and growth in EMT6/Ro multicellular tumour spheroids
Authors: Casciari J. J. , Sotirchos S. V. , Sutherland R. M.
Published: May 2008

Abstract. In order to determine the role of micromilieu in tumour spheroid growth, a mathematical model was developed to predict EMT6/Ro spheroid growth and microenvironment based upon numerical solution of the diffusion/reaction equation for oxygen, glucose, lactate ion, carbon dioxide, bicarbonate ion, chlorine ion and hydrogen ion along with the equation of electroneutrality. This model takes into account the effects of oxygen concentration, glucose concentration and extracellular pH on cell growth and metabolism. Since independent measurements of EMT6/Ro single cell growth and metabolic rates, spheroid diffusion constants, and spinner flask mass transfer coefficients are available, model predictions using these parameters were compared with published data on EMT6/Ro spheroid growth and micro‐environment. The model predictions of reduced spheroid growth due to reduced cell growth rates and cell shedding fit experimental spheroid growth data below 700 μm, but overestimated the spheroid growth rate at larger diameters. Predicted viable rim thicknesses based on predicted near zero glucose concentrations fit published viable rim thickness data for 1000 μm spheroids grown at medium glucose concentrations of 5.5 mM or less. However, the model did not accurately predict the onset of necrosis. Moreover, the model could not predict the observed decreases in oxygen and glucose metabolism seen in spheroids with time, nor could it predict the observed growth plateau. This suggests that other unknown factors, such as inhibitors or cell‐cell contact effects, must also be important in affecting spheroid growth and cellular metabolism.
Chemical protection and cell‐cycle effects on radiation‐induced mutagenesis
Authors: Grdina D. J. , Sigdestad C. P.
Published: May 2008

Abstract. Chinese hamster ovary cells in the exponential phase of growth were harvested and separated by the method of centrifugal elutriation into subpopulations enriched with up to 95% G1 phase, 70% S phase and 65% G2+ M phase cells. Cell cycle distributions were routinely monitored by flow cytometry. Following elutriation, aliquots of cells from each of the enriched cell fractions were incubated in the presence or absence of 4 MM of 2–[(aminopropyl)amino] ethanethiol (WR‐1065) for 30 min at 37°C. The cells were then irradiated with 60Co γ‐rays or fission‐spectrum neutrons from the JANUS research reactor. Both cell killing and mutagenesis were determined. Regardless of the radiation quality used, cells enriched in G1 phase were the most sensitive to radiation‐induced mutagenesis at the hypoxanthine‐guanine phos‐phoribosyl transferase locus. The relative magnitude of protection exerted by WR‐1065 differed for each of the elutriator separated cell populations. The greatest magnitude of protection, however, was observed for G1‐enriched populations, regardless of the radiation quality used or the biological end‐point tested.
A flow cytometric study of the effect of heat on the kinetics of cell proliferation of Chinese hamster V‐79 cells
Authors: Ormerod M. G. , Imrie P. R. , Loverock P. , Haar G. Ter
Published: May 2008

Abstract. Two methods involving labelling cells with bromodeoxyuridine (BrdUrd) have been used to study by flow cytometry the effect of hyperthermia (43°C for up to 1 h) on Chinese hamster V79 cells. One method involved the use of an antibody to BrdUrd after pulse‐labelling the cells either before or at time intervals after treatment. In the second method, the cells were incubated continuously in BrdUrd after heat treatment, and the components of the cell cycle were then visualized by staining with a combination of a bis‐bcnzimidazole and ethidium bromide. All three methods showed that heating at 43°C stopped DNA synthesis which, at 37°C, subsequently recovered reaching the normal rate 8–12 h later. The cells in S phase at the time of treatment then progressed to G2 where they were further delayed. Cells heated in G1. after the recommencement of synthesis, progressed around the cycle, albeit slower than in unheated cells. The difference between the cells in G1 and S phases at the time of treatment may account for the greater sensitivity of S phase cells to hyperthermia.
Altered proliferative kinetics in PHA‐activated human T‐lymphocytes treated with the anti‐HLA class I monoclonal antibody 01.65
Authors: Rubini M. , Selvatici R. , Orlando P. , Balugani S. , Balboni A. , Gandini E.
Published: May 2008

Abstract. Anti‐HLA class I monoclonal antibody (mAb) 01.65 inhibited phyto‐haemagglutinin (PHA)‐induced human lymphocyte proliferation. The inhibitory effect was inversely correlated to the strength of the proliferative response. It was increased when lymphocytes were stimulated with suboptimal doses of PHA but it disappeared with supraoptimal doses. Proliferation inhibition was achieved by prolonging the cell cycle time and by slowing down its recruitment rate. The former effect was not restricted to the G1‐phase but also included the S phase. These results support the idea that HLA class I molecules are important in the PHA‐induced proliferation of human T‐lymphocytes.
Proliferative changes in two murine tumours in response to single or fractionated doses of X‐rays
Authors: Wilson G. D. , Martindale C. A. , Soranson J. A. , Carl U. M. , McNally N. J.
Published: May 2008

Abstract. Studies were carried out to investigate proliferative changes in two murine experimental tumours in response to radiation. Results were generated using bro‐modeoxyuridine labelling and flow cytometry. This study demonstrates the possible ambiguity of previous studies using tritiated thymidine in which inability to discriminate normal and tumour cell components in murine tumours may lead to different values for cell kinetic parameters. In particular, the sarcoma F appeared to have a growth fraction of 0.62 when all cells were considered; in reality the growth fraction of the tumour cells only (based on DNA content discrimination) was close to unity. Radiation, administered either as single or fractionated doses, caused little change in the proliferative characteristics of the sarcoma F tumour but had profound effects on the adenocarcinoma Rhodesia tumour. Major changes were the accumulation of cells in G2 for several days after the end of the radiation treatment in both tumours and a dramatic drop in labelling index of the Rhodesia tumour. In neither tumour was there any evidence to suggest an increase in tumour cell proliferation during or after the irradiations. The diploid cells within the sarcoma F tumour showed an initial depression of labelling index followed by a rapid increase overshooting the control labelling index at higher radiation doses. Much of the effects could be attributed to cell cycle delays.
The role of cell death in the growth of preneoplastic lesions: a Monte Carlo simulation model
Authors: Berman J. J. , Moore G. W.
Published: May 2008

Abstract. A variety of experimental and clinical examples of preneoplasia demonstrate that regression of early lesions is common. This paper examines the hypothesis that early lesions operate under the identical growth kinetics of ‘late’ lesions (neoplasms), but that kinetic features favouring continuous growth in established lesions tend to favour extinction of lesions composed of small numbers of cells. Growth simulations of early lesions were produced using the Monte Carlo method, a technique demanding intensive computations. With the advent of powerful personal computers, this technique is now widely available to biologists. Simulating growth under conditions of cell loss similar to those observed in established tumours, the model predicts that the great majority of initiated cell clusters are expected to reach extinction within a few cell doubling times, and most early (promoted) lesions would not likely progress to the size of a clinically detectable lesion within the life span of the host organism. These Monte Carlo simulations provide a model of initiated cell growth consistent with the recently demonstrated role of early lesion cell death in the development of human lymphomas and in transgenic mice expressing the bcl‐2 oncogene. The model demonstrates that small increments in the intrinsic cell loss probability in even the earliest progenitors of malignancy can strongly influence the subsequent development of neoplasia from initiated foci.
A model of the control of cellular regeneration in the intestinal crypt after perturbation based solely on local stem cell regulation
Authors: Paulus U. , Potten C. S. , Loeffler M.
Published: May 2008

Abstract. The control mechanisms involved in regeneration of murine intestinal crypts after perturbations are presently not well understood. The existence of some feedback signals from the cells on the villus to the cells in the crypt has been suggested. However, some recent experimental data point to the fact that regeneration in the crypt starts very early after perturbation, at a time when the villus cell population has hardly changed. In particular, this early cell proliferative activity is seen specifically at the bottom of the crypt, i.e. in the presumed stem cell zone and furthest from the villus.

The objective of this study was to investigate whether a new concept of regulation operating solely at the stem cell level could explain the present mass of accumulated data on the post‐irradiation recovery, which is an extensively studied perturbation from the experimental point of view. In order to check its validity, the new concept was formalized as a mathematical simulation model thus enabling comparison with experimental data. The model describes the cellular development from stem cells to the mature villus cells. As a basic feature it is assumed that the self‐maintenance and the cell cycle activity of the stem cells are controlled by the number of these cells in an autoregulatory fashion. The essential features of the experimental data (i.e. the recovery with time and the consistency between different types of measurements) can be very well reproduced by simulations using a range of model parameters. Thus, we conclude that stem cell autoregulation is a valid concept which could replace the villus crypt feedback concept in explaining the early changes after irradiation when the damage primarily affects the crypt. The question of the detailed nature of the control process requires further investigation.
Ferritin‐iron increases killing of Chinese hamster ovary cells by X‐irradiation
Authors: Nelson J. M. , Stevens R. G.
Published: May 2008

Abstract. Stationary‐phase Chinese hamster ovary cells were cultured in medium containing ferritin (‐19% iron by weight) added at concentrations ranging from 0 to 128 μg/ml. One set of cultures was unirradiated, and another set was exposed to 4.0 Gy of X‐ray. Clonogenic cell survival was assessed in each set of cultures. In the absence of added ferritin, 4.0 Gy killed approximately 50% of the cells. In the absence of radiation, ferritin was not toxic at less than 48 μg/ml; above 48 μg/ml, toxicity increased with concentration. Apoferritin was not toxic at any concentration tested (up to 1000 μg/ml). Although 32 μg/ml ferritin, reflecting only a 3–6 fold increase in iron concentration over normal serum, was not toxic, it reduced the survival of X‐irradiated cells by an additional 75%. These results indicate that a sublethal concentration of ferritin can be a potent radiosensitizer. This suggests the possibility that high body iron stores may increase susceptibility to radiation injury in humans.
Autoradiographic studies of rat astroglial cell proliferation in vitro with and without treatment with basic fibroblast growth factor
Authors: Korr H. , Siewert E. , Bertram C. , Labourdette G. , Sensenbrenner M.
Published: May 2008

Abstract. Using specific autoradiographic methods, cell cycle parameters of untreated and basic fibroblast growth factor (bFGF)‐treated astroglial cells from newborn rats grown in primary culture were directly measured. The mode of proliferation was also analysed. In untreated cultures, S phase duration (Ts= 6.9–13.1 h) and cell cycle time (Tc= 10–18 h) can be modified by about a factor of 2 depending on the culture conditions (serum‐supplemented or defined medium, thyroid hormone concentration). However, growth fraction (GF = 0.15) and the ratio Ts/Tc remain stable. With increasing days in vitro (DIV) (DIV 7‐DIV 20), Ts (7.8–10.6 h) and Tc (10–21 h) are prolonged and GF (0.14–0.06) decreases, probably due to cell maturation. In general, astroglial cells proliferate exponentially with a GF < 1, but stop proliferating about 30–36 h after the last feeding, probably caused by exhaustion of the medium. However, after refeeding they continue to proliferate. As opposed to in vivo, no transition of non‐proliferating cells into the GF occurs. After addition of bFGF, GF increases (e.g. GF at DIV 7 = 0.43), but Ts and Tc are not influenced at DIV 7 and 12. At DIV 20, bFGF additionally shortens Ts and Tc, thereby producing values of Ts, Tc and GF like ‘younger’ cultures. However, the revitalizing effect on ‘mature’ cells is only transitory. In general, bFGF leads to a single re‐entry of Go cells into the GF. Thereafter, bFGF does not affect the mode of proliferation.
Rabbit embryo‐fetal fluid decreases the cell cycle activities of DU‐145 cells
Authors: Lambert R. D. , Dufour M. , Blouin L.
Published: May 2008

Abstract. Successful reproduction requires tight control of cell proliferation and differentiation. Rabbit blastocoelic fluid contains such regulatory factors. For instance, it inhibits tumour or transformed cell proliferation. In this study, DU‐145 cells have been used to characterize further this inhibitory activity. Maximal inhibition of cell proliferation is observed at day 12 of embryo‐fetal development and this is accompanied by a strong reduction of [3H]‐thymidine incorporation. DNA specific staining and analysis by flow cytometry show that cells are not stopped at any specific stage of the cell cycle. Using bromodeoxyuridine incorporation in combination with pro‐pidium iodide labelling, it has been possible to estimate the percentage of labelled cells, the duration of the S phase of the cell cycle derived from their relative movement and also the proportion of cells participating to the cell cycle. In the presence of embryonic and fetal fluids collected on day 12 (EFF D‐12) the duration of the S phase and the doubling time are considerably increased and the percentage of cells participating in the cell cycle is decreased. The results also show that treatment with EFF D‐12 induces the release of the cells from the monolayer. Taken altogether, these results suggest that EFF D‐12 increases the duration of the cell cycle. This reduction of the mitotic activities lead up to cell death with subsequent release of cells into the culture medium.
Humoral stimulating activities in post‐cyclophosphamide rat sera and their purified fractions
Authors: Pujic N. , Hegedis Lj. , Pavelic K. , Casl T. , Marusic S. , Savovski K. , Dujic A. , Dimitrijevic B.
Published: May 2008

Abstract. Enhanced humoral, stimulating activities (HSAs) of post‐cyclophosphamide (CY) sera and their purified fractions, acting on mitogen activated T‐lymphocytes, were detected in Wistar rats after treatment by high single doses of the aplasia producing alkylating cytostatic drug CY. These activities, monitored in vitro, were partially purified from post‐CY sera, collected 2, 4, and 7 days after treatment, yielding fractions with higher humoral stimulating activity. The preliminary purification included molecular cutting by Amicon‐Diaflo filters (1‐30 kDa pore size range) and gel‐filtration on Sephadex G‐50 and G‐75. Results show that post‐CY sera partially purified fraction (10–20 kDa), enhances the proliferation of hydrocortisone resistant (HCR) T‐lymphocytes up to threefold under microculture conditions (P«0.001); partially purified post‐CY sera fraction (10–20 kDa) increases the proliferative response of T‐cells in microcultures to the mitogenic 145–2C11 monoclonal antibody (mAb) up to tenfold (P«0.001). These results show that the activity of stimulating factors is localized in the molecular weight range of 10–20 kDa.
Cell cycle progression in human cells following re‐oxygenation after extreme hypoxia: consequences concerning initiation of DNA synthesis
Authors: Åmellem Ø. , Pettersen E. O.
Published: May 2008

Abstract. The initiation of DNA synthesis and further cell cycle progression in cells during and following exposure to extremely hypoxic conditions in either G1 or G2+M has been studied in human NHIK 3025 cells. Populations of cells, synchronized by mitotic selection, were rendered extremely hypoxic (< 4 p.p.m. O2) for up to 24n h. Cell cycle progression was studied from flow cytometric DNA recordings. No accumulation of DNA was found to take place during extreme hypoxia. Cells initially in G1 at the onset of treatment did not enter S during up to 24 h exposure to extreme hypoxia, but started DNA synthesis in a highly synchronous manner within 1.5 to 2.25 h after reoxygenation. The duration of S phase was only slightly affected (increased by ≅10%) by the hypoxic treatment. This suggests that the DNA synthesizing machinery either remains intact during hypoxia or is rapidly restored after reoxygenation. Cells initially in G2 at the onset of hypoxia were able to complete mitosis, but further cell cycle progression was blocked in the subsequent G^ Following reoxygenation, these cells progressed into S phase, but the initiation of DNA synthesis was delayed for a period corresponding to at least the duration of normal G1 and did not appear in a synchronous manner. In fact, cell cycle variability was found to be increased rather than decreased as a result of exposure to hypoxia starting in G2. We interpret these findings as an indication that important steps in the preparation for initiation of DNA synthesis take place before mitosis. Furthermore, the change in cell cycle duration induced by hypoxia commencing in G1 is of a nature other than that induced by hypoxia commencing in other parts of the cell cycle.
Induction of replicative senescence by 5‐azacytidine: fundamental cell kinetic differences between human diploid fibroblasts and NIH‐3T3 cells
Authors: Weller E. M. , Poot M. , Hoehn H.
Published: May 2008

Abstract. To analyse the putative role of methylation of cytosine residues in the nuclear DNA as a regulatory step during cellular ageing, we incubated ageing human amniotic fluid derived fibroblast‐like cells and non‐ageing NIH‐3T3 cells with 5‐azacytidine. BrdUrd/Hoechst and acridine orange (AO) flow cytometry was used to compare the effects of the base analogue on cell proliferation and cell differentiation. In NIH‐3T3 cultures, 96 h exposures to 4 μM 5‐azacytidine caused diminished cell proliferation due to cell arrest in the G1 compartments of the second and third cell cycles of serum stimulated cells. The exit from the G0/G1 compartment was not affected. The 5‐azacytidine induced cell kinetic disturbances were unstable in NIH‐3T3 cultures, such that pre‐treated cells reverted to normal cell cycle transit within 2–3 days after termination of treatment. In contrast, 5‐azacytidine pre‐treated amniotic fluid derived fibroblast‐like cell cultures showed persistently elevated G2 phase arrests and delayed G0/G1 phase exit kinetics, which explain the premature cessation of proliferation observed in these primary cultures. In both cell systems, 5‐azacytidine exposed cultures showed elevated numbers of G1 phase cells with increased RNA content as revealed by AO flow cytometry. Again, this effect was reversible in NIH‐3T3 cells but not in amniotic fluid derived fibroblast‐like cells. These contrasting responses to 5‐azacytidine are likely to reflect intrinsic differences in methylation patterns or de novo methylase activity between ageing cell strains and non‐ageing cell lines.
Inhibition of distinct steps in the adipocyte differentiation pathway in 3T3 T mesenchymal stem cells by dimethyl sulphoxide (DMSO)
Authors: Wang H. , Scott R. E.
Published: May 2008

Abstract. The process of adipocyte differentiation in murine 3T3 T mesenchymal stem cells involves three well‐defined steps: 1 predifferentiation growth arrest; 2 nonterminal (reversible) differentiation and 3 terminal differentiation associated with the irreversible loss of proliferative potential. To further investigate these processes, the effects of dimethyl sulphoxide (DMSO), an agent that affects differentiation in several other cell systems, was tested. The results show that DMSO modulates two distinct steps of adipocyte differentiation. The first effect is evident when growing 3T3 T cells are cultured in differentiation‐inducing medium in the presence of DMSO. Therein the expression of adipocyte phenotype is inhibited because the cells fail to growtharrest at the predifferentiation growth arrest state. Instead in the presence of DMSO, cells growth‐arrest at a biological state that does not support differentiation. The second effect is evident if nonterminally differentiated adipocytes are cultured in terminal differentiation‐inducing medium containing DMSO. Therein the terminal step in differentiation is inhibited. These inhibitory effects occur in a dosage‐dependent manner; maximum inhibition of differentiation requires 2% DMSO. Therefore, whereas DMSO typically promotes differentiation in other cell systems, DMSO inhibits multiple steps in the process of adipocyte differentiation. These observations support the conclusion that a single pharmacological agent can have markedly different effects on specific cell types. Even more important, the data establish that DMSO can now be used as a tool to study the molecular mechanisms involved in the multistep process of adipocyte differentiation.
Ki‐67 expression and BrdUrd incorporation as markers of proliferative activity in human prostate tumour models
Authors: Van Weerden W. M. , Moerings E. P. C. M. , Kreuningen A. van , De Jong F. H. , Van Steenbrugge G. J. , Schröder F. H.
Published: May 2008

Abstract. The validity of the use of the monoclonal antibodies Ki‐67 and anti‐BrdUrd to evaluate proliferative activity of human prostate tumour models was studied. Growth of the transplantable PC‐82 and PC‐EW prostate tumours, as assessed by tumour volume measurements, was significantly correlated with the proliferative activity as reflected by BrdUrd incorporation into DNA (r= 0.64 and r= 0.78, respectively). The proliferative activity of PC‐82 tumours detected by Ki‐67 antigen expression paralleled the pattern observed with BrdUrd (r= 0.51) and a significant correlation (r= 0.60) between the results obtained with both markers was found. In growing PC‐82 and PC‐EW tumours only small variations in the Ki‐67 and BrdUrd indices were observed. In contrast, Ki‐67 expression in regressing PC‐82 tumours varied considerably (2.7 ± 2.2%). The BrdUrd index in regressing PC‐32 tumours showed less variation (1.3 ± 0.2%), but part of the BrdUrd‐positive cells were found in the stromal (murine) part of the regressing tissue. It is concluded that the Ki‐67 and BrdUrd proliferation markers are reliable parameters to monitor changes in growth of prostate tumour lines, but that in slow growing or regressing tumours Ki‐67 and BrdUrd data should be interpreted with caution.
Multiparameter flow cytometric analysis of the effects of indomethacin on adipocyte differentiation in A31T6 cells
Authors: Smyth M. J. , Wharton W.
Published: May 2008

Abstract. A31T6 proadipocytes, derived from BALB/C‐3T3 clone A31, develop responsiveness to differentiation‐promoting agents at density‐arrest and differentiate into adipocytes, as determined by the accumulation of cytoplasmic lipid droplets. A flow cytometric assay is being employed to monitor the acquisition of aspects of the differentiated phenotype. In this study, the assay is used to monitor both the rate of differentiation, as defined by the appearance of cells containing lipid droplets and the rate of adipocyte maturation, which involves measurement of increases in cytoplasmic lipid in cells already committed to the differentiation programme. Specifically, we show that 1 treatment with a combination of indomethacin and dexamethasone causes the maximum percentage differentiation in the population, 2 addition of indomethacin in combination with either dexamethasone or insulin increases the rate of differentiation, and 3 indomethacin selectively increases the maturation of adipocytes, measured as an increase in the amount of lipid per cell. The cytometric assay used in these experiments has allowed determination of the effects of indomethacin on aspects of the adipocyte phenotype that cannot be measured by standard techniques.
Biochemical and morphological characterizations of DU‐145 cell mortality in rabbit embryo‐fetal fluid
Authors: Lapointe J. , Bergeron D. , Dufour M. , Dubé D. , Govindan M. V. , Lambert R. D.
Published: May 2008

Abstract. Rabbit embryo‐fetal fluid (EFF) contains regulatory factors of cell proliferation which increase the duration of the cell cycle, induce a quiescent status in some cells and lead up to cell death in others. The objective of this study was to demonstrate which of the two processes, namely necrosis or apoptosis, was responsible for the cell death. Inhibitors of protein synthesis, and nuclease and phospholipase A2 activities did not restore the viability of the cells treated with EFF. Using a combination of DNA labelling and extraction, it was possible to show that a large proportion of DNA was fragmented in the cells released in the supernatant while only a very small portion of DNA was fragmented in the monolayer cells. EFF did not induce fragmentation of DNA into nucleosome‐sized subunits as analysed using polyacrylamide gel electrophoresis. Nevertheless, using cytofluorometric analysis, it was possible to demonstrate that 50% of the cells released in the supernatant contained a lower quantity of DNA per cell than in the control cells. This was also observed with EFF‐treated monolayer cells but not in the control monolayer cells. The reduction of the DNA content per monolayer cell became significant at 48 h of treatment with EFF. Electron microscopic analysis did not reveal blebbing of the cells. However, depletion of glycogen, condensation of mitochondria and increasing number of lysosomes and residual bodies were observed upon treatment with EFF. From these experiments we conclude that the DU‐145 cells treated with EFF do not die by apoptosis, but rather seem to die by necrosis.
Hydroxyurea exposure alters mouse testicular kinetics and sperm chromatin structure
Authors: Evenson D. P. , Jost L. K.
Published: May 2008

Abstract. The effects of hydroxyurea (HU) on testicular cell kinetics and sperm chromatin differentiation were investigated in mice. Whole testis, minced testicular cell suspensions and caudal epididymal sperm cells were obtained at 8 and 29 days after i.p. injections containing 0, 25, 50, 100, 200, 400 and 500 mg/kg HU X 5 days. Testis weights were unaffected by 25 mg/kg HU while 500 mg/kg caused up to a 50% loss of testicular weight by 29 days. Flow cytometrically measured acridine‐orange (AO) stained testicular cells revealed altered population ratios at the highest dosages at 8 days and for all dosages except 25 mg/kg HU at 29 days. At 8 days, 400–500 mg/kg HU caused a near depletion of tetraploid cells. Flow cytometry of AO stained sperm, previously treated with acid to potentially induce DNA denaturation, was used to follow the shift from normal chromatin structure to an abnormal form with increased sensitivity to DNA denaturation in situ. The extent of DNA denaturation was quantitated for each cell by the computer‐derived value alpha t, αt= [red/(red+green) fluorescence]. The flow cytometry measures, standard deviation of αt (SDαt), mean of αt (Xαt) and cells outside the main peak of αt (COMPαt), gave similar dose response curves to the sperm head morphology assay. SDαt was more sensitive than the Xαt as a measure of HU‐induced alteration of chromatin structure.

The major conclusions reached are that HU inhibits DNA synthesis, probably by inhibiting ribonucleotide reductase, causing maturation depletion of pachytene spermatocytes and, subsequently, depletion of meiotic daughter cells and differentiated cell types leading to mature sperm. This inhibition of DNA synthesis is related to an alteration of sperm chromatin structure and abnormal sperm head morphology.
Accumulation of anchorage independent cells showing amplified genes (CAD) during thein vitropropagation of CHEF18 Chinese hamster cells
Authors: Vatteroni L. , Piras A. , Mariani T. , Caligo M. A. , Rainaldi G.
Published: May 2008

Abstract.Anchorage independence and gene amplification have frequently been associated with a transformed or tumorigenic phenotype in cultured mammalian cells. However, it is unknown whether these two traits occur as related events during transformation, or are independent features of the transformed phenotype. To clarify this point, immortalized, untransformed CHEF18 Chinese hamster cells were propagated in culture until they became transformed and tumorigenic. The frequencies with which CHEF18 cells formed colonies either in soft agar, in medium containing N‐phosphonacetyl‐L‐asparate or in the two selective media simultaneously, were determined. The results indicate that anchorage independence and CAD gene amplification spontaneously arose during the propagation of the cells and that their concurrent emergence was not the consequence of independent events. However, the kinetics of their appearance suggests that anchorage independence is the early event whereas gene amplification might represent one of the numerous events which can be dynamically selected in anchorage‐independent cells.
Pre‐irradiation of tissue culture flasks leads to diminished stem and progenitor cell production in long‐term bone marrow cultures
Authors: Rooney P. , Wright E.G.
Published: May 2008

Abstract. Empty plastic tissue culture flasks were exposed to X‐irradiation doses of 0.3–10.0 Gy, prior to the establishment of long‐term bone marrow cultures. During the course of a 10 week culture period, all irradiated plastic flasks exhibited a dramatic decrease in the number of both haemopoietic stem cells and myeloid progenitor cells, in the non‐adherent layer, when compared with controls. This decrease was not due to a decrease in the number of non‐adherent cells produced. Histological examination of non‐adherent cells showed an increase in mature granulocytic cells with few blast cells. Morphologically, the adherent layers of irradiated flasks demonstrated a delay in appearance or absence of fat cell production. X‐irradiation of glass tissue culture flasks had no deleterious effect.
Cell proliferation kinetics of the bile duct epithelium of the rat
Authors: Koike N. , Saitoh K. , Todoroki T. , Kawamoto T. , Iwasaki Y. , Nakamura K.
Published: May 2008

Abstract. Cell proliferation kinetics of the extrahepatic bile duct were studied by flash and cumulative labelling methods and immunohistochemical techniques. We compared the cell kinetics of the epithelium of the intra‐ and extra‐pancreatic bile ducts and of the bile duct of the ampulla in rats administered intraperitoneally with bromodeoxyuridine (BrdUrd). After a single injection of BrdUrd (flash labelling), labelled cells appeared in the lower portion of the downgrowths of the epithelium in the intra‐and extra‐pancreatic bile ducts. A gradual accumulation of the labelled cells at the surface epithelium was observed during the cumulative labelling. After cumulative labelling the labelled cells gradually decreased in number and were finally confined to the degenerative cell zone of the surface epithelium 30 days later. Similarly, after a single injection of BrdUrd, the labelled cells in the bile duct of the ampulla appeared at the lower half of the crypt from where they migrated to the upper portion during cumulative labelling. These findings indicate that epithelial cells of the bile duct are renewed at the lower portion of the downgrowths of the epithelium, or crypt, and shed from the surface epithelium or upper portion of the fold. The labelling indices reached 23.83 ± 7.47% in the intra‐pancreatic bile duct, 14.74 ± 7.99% in the extra‐pancreatic bile duct and 43.42 ± 4.40% in the bile duct of the ampulla at the end of 70 h cumulative labelling. The fluctuating values of the labelling index were higher in the bile duct of the ampulla than in the intra‐ or extra‐pancreatic bile ducts. These results indicate that the bile‐duct epithelium undergoes a slower renewal rate than the other parts of the gastrointestinal tract, and that the renewal time of the epithelial cells is shorter at the bile duct of the ampulla than at the intra‐ or extra‐pancreatic bile ducts.
Restriction endonuclease recognition and Southern hybridization of bromodeoxyuridine‐substituted genomic DNA
Authors: Asai A. , Hirai H. , Bodell W. J. , Hoshino T.
Published: May 2008

Abstract. Human glioma cell lines exposed to various concentrations of bromode‐oxyuridine (BrdUrd) were studied to determine the effect of BrdUrd substitution on restriction endonuclease recognition and Southern hybridization of genomic DNA. BrdUrd substitution had no effect on the recognition of restriction endonucleases. When the exposure to BrdUrd was 2 h or less and the BrdUrd substitution rate was less than 40%, there was no difference in the density of hybridized bands after Southern hybridization using human non‐recombinant complementary DNA as a probe. Hybridization was suppressed significantly by exposures longer than 24 h or BrdUrd substitution rates greater than 40%. These results suggest that the BrdUrd substitution rate and the exposure time to BrdUrd influence the hybridization reaction by a DNA probe. Brief exposure (up to 2 h) to BrdUrd does not influence restriction endonuclease recognition or Southern hybridization of genomic DNA.
Induction of interleukin‐2 receptor α‐chain gene expression by cytochalasin B and 12‐O‐tetradecanoylphorbol‐13‐acetate in T lymphocytes
Authors: Gunes H. , Mastro A. M.
Published: May 2008

Abstract. The high affinity form of interleukin‐2 receptor (IL‐2R) is composed of two subunits; the α (p55) and β (p75). The α chain, unlike the β, is expressed only on activated T lymphocytes. Therefore, high affinity binding of interleukin‐2 (IL‐2) is controlled by the expression of the IL‐2R α‐chain. In this study, we examined the effect of cytochalasin B (CB) plus 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) on expression of IL‐2 and IL‐2R. Northern blot and flow cytometric analysis showed that the IL‐2R α‐chain was expressed both at mRNA and protein levels. However, IL‐2 gene expression was not induced by this treatment. Unlike the cells treated individually with CB or TPA, cells treated with CB plus TPA accumulated IL‐2R mRNA at all the times examined. In order to determine the percentage of cells that incorporated tritiated thymidine ([3H]dT) in the presence of IL‐2 after treatment with CB plus TPA, autoradiography was carried out. We found that about 11% of the cells were labelled. Because the percentage of labelled cells and cells expressing IL‐2R α‐chain was relatively low (11% and 9% respectively), perhaps CB plus TPA caused IL‐2R expression in only a subset of T cells.
Growth slow‐down and growth arrest of human colon carcinoma cells HCT‐8 in vitro after exposure to 5‐fluoro‐2′‐deoxyuridine
Authors: Malmberg M. , Slocum H. K. , Rustum Y. M.
Published: May 2008

Abstract. Cellular heterogeneity in drug response denotes a mixed response among individual cells in a drug treated population. Individual cell responses may be more complex than ‘cell kill’and ‘no response’. In this study we employed a colony formation assay and high‐resolution image analysis to detect the various responses such as immediate and delayed cessation of growth, growth delay and growth slow‐down, at the level of the individual colony. The evaluation was carried out using a human ileocaecal adenocarcinoma cell line (HCT‐8) and the anti‐tumour agent 5‐fluoro‐2′‐deoxyuridine (FdUrd). In the presence of a drug concentration which, in standard monolayer assays, inhibits the growth to about 50% (IC50) only about 20% of the colonies ceased to grow and the remaining colonies grew at a growth rate of about 70% of control. At an FdUrd concentration which, in standard monolayer assays, reduced growth by >90% (>IC90), about 50% of the cells grew, with growth rates of about 30% of control. The slowing of growth, most prominent at lower drug concentrations, should be considered in determining mechanisms of drug action at the individual cell level. In clinical situations in which high drug doses are precluded by toxicity to normal tissues, growth slow‐down may play a significant role in tumour response.
Comparison of isotopic and immunohistochemical methods of studying epithelial cell proliferation in hamster tongue
Authors: Warnakulasuriya K. A. A. S. , Johnson N. W.
Published: May 2008

Our purpose was to validate different approaches to the study of cell proliferation in stratified squamous epithelia, using oral mucosa as a model. Dorsal and ventral tongue from the hamster were examined following in vivo labelling with tritiated thymidine and bromodeoxyuridine (BrdUrd), and in vitro labelling with BrdUrd. These were compared with direct immunolabelling of fixed tissue sections with monoclonal antibody PC10. For the former methods S phase cells were quantified following autoradiography or immunohistochemistry. We conclude that the proliferative status of simple, flat, lining mucosae such as ventral tongue can be derived by all three prelabelling methods and, on average, 18–19 cells per surface millimetre length were in DNA synthesis. On the other hand dorsal tongue epithelium, which is thicker, has an undulating morphology and a complex cell renewal pattern, gives different results with the three labelling methods. In both sites the proliferating cell nuclear antigen (PCNA) index was fourfold that obtained by nucleotide labelling. This is consistent with PCNA marking proliferative cells in other phases of the cell cycle in addition to the S phase. Thus, there are potential differences between the information on proliferative status derived by PCNA immunohistochemistry and other established cell cycle markers, which need to be taken into account in the interpretation of epithelial cell kinetic data in health and disease.
Binucleation and polyploidization patterns in developmental and regenerative rat liver growth
Authors: Gerlyng P. , Åbyholm A. , Grotmol T. , Erikstein B. , Huitfeldt H. S. , Stokke T. , Seglen P. O.
Published: May 2008

The hepatocellular binucleation rate, measured as the percentage of binuclear cells amongst newly formed bromodeoxyuridine‐labelled and immunostained collage‐nase‐isolated rat hepatocytes, decreased from 12% to 4% between days 30 and 40 after birth, rose to 20% between days 50 and 60, and then declined again to the adult rate of about 10% at day 80. During regenerative growth following a two‐thirds partial hepatectomy, the rate of binucleation declined to about 3%, causing the fraction of binuclear cells to fall from 27% (before hepactectomy) to 5% (at 45 h after hepactectomy) as pre‐existing binuclear cells replicated and formed mononuclear daughter cells. Essentially all (97%) hepatocytes replicated at least once, starting their DNA synthesis at around 13 h and reaching a peak at 30 h, irrespective of ploidy and nuclearity. At later time points, the diploid hepatocytes had a higher labelling index than the polyploid cells, suggesting a greater tendency to go through several cell cycles.
Cell kinetic characterization of the epidermal growth factor dependent BALB/MK line using flow cytometric analysis of DNA content and iododeoxyuridine incorporation
Authors: Van Hooijdonk C. A. E. M. , Van Erp P. E. J. , Freund R. L. , De Jongh G. J. , Schalkwijk J. , Van De Kerkhof P. C. M. , Mier P. D.
Published: May 2008

Epidermal hyperproliferation (psoriasis, wound repair) is the result of quiescent (G0) keratinocytes being recruited into the cell cycle. A detailed characterization of the cell cycle kinetic parameters of the mouse keratinocyte line (Balb/MK) has been carried out with the aid of bivariate iododeoxyuridine (IdUrd) and DNA analysis using flow cytometry, in order to establish whether it might provide a useful model for the study of the biochemical events controlling recruitment into the cell cycle. Balb/MK keratinocytes were cultured using low Ca2+ Dulbecco's modified Eagle's medium/F12 in the presence of 10% dialysed fetal bovine serum and 4 ng/ml epidermal growth factor (EGF). IdUrd labelling followed by flow cytometric analysis of trypsinized cells showed that about 95% of the population were actively cycling, with a cell cycle time of around 14 h and no significant contact inhibition. Omission of serum and EGF followed by IdUrd pulse‐labelling indicated that the cells progressively withdrew from the cycle and, after 16h, less than 10% incorporated IdUrd. Subsequent restimulation with serum resulted in a synchronized cohort of cells being recruited. Entry into the S phase of the cell cycle (IdUrd incorporation) started at 8 h and was maximal between 12 h and 16 h after stimulation. Restimulation with EGF alone reached a growth fraction of 87% after 24 h continuous labelling compared with 97% using serum together with EGF. Epidermal growth factor already showed a significant stimulation at 10 pg/ml compared with the controls. There is a broad plateau centred on 5 ng/ml, followed by a slight decline above this level. We conclude that the use of a cell line with defined cell cycle kinetic parameters which can be switched between the quiescent and cycling states in a fully defined medium will provide an ideal model for biochemical studies of the relevant signal transduction pathways in keratinocytes.