bluuebunny/crossref_metadata_2025 huggingface dataset:
Streaming kidney
Authors: Zajicek G. , Arber N.
Published: May 2008

Abstract. Twenty‐five male young adult random bred rats, weighing 250–300 g, were injected with 18·5 kBq/g body weight tritiated thymidine (specific activity 185 GBq/mM). The rats were then killed in groups of five, at the following times: 1 h and14, 30, 60 and 120 days. The kidneys were processed histologically and dipped into liquid emulsion, exposed for 3 weeks and developed. Kinetic measurements were restricted to juxta‐medullary glomeruli, their adjacent convoluted tubules and medullary tubuli. All other nephrons were ignored. The medullary pole of the juxta‐medullary glomerulus served as reference point for all measurements and was referred to as the origin. The distance of a labelled cell from the origin was measured with an eye‐piece micrometer and expressed in terms of two units: distance (μm) and cell location, defined as the number of cells separating a labelled cell from origin. Since only medullary nephrons were considered, these measurements represent distances directed toward the papilla. One hour after labelling, most cells were in the vicinity of the juxta‐medullary glomerulus, not further than 800 μm from the origin in the direction of the papilla. During the following days labelled cells advanced toward the papilla at a daily velocity of 13.8 pm, covering 1.1 locations/day. Kinetically, the juxta‐medullary nephron is a two‐compartment cell renewal system. Its compartments, the progenitor (P) and the functional (a), cover locations 0–80 and 81–150, respectively. The first feeds the second with cells. Morphologically, the progenitor compartment includes proximal and distal convoluted epithelia and a part of thick straight tubules. Other nephron portions belong to the Q‐compartment. It is assumed that the nephrocyte is a part of a cell stream directed toward the papilla, which probably also includes stroma and vasculature.
Evaluation of glucocorticoid‐induced DNA fragmentation in mouse thymocytes by flow cytometry
Authors: Telford W. G. , King L. E. , Fraker P. J.
Published: May 2008

Abstract. The ability of glucocorticoids to induce apoptosis or programmed cell death in mouse thymocytes is well‐established. Measurement of apoptosis‐associated internucleosomal DNA fragmentation through determination of the percentage of fragmented DNA by electrophoresis or centrifugation of whole cell lysates is by far the most common means of quantifying apoptosis. Since these methods measure DNA fragmentation in whole cell lysates rather than intact cells, they have severe limitations, particularly with heterogeneous cell populations. When mouse thymocytes were incubated with glucocorticoids, fixed, stained with propidium iodide and analysed flow cytometrically for cell cycle distribution, a distinct subpopulation of cells was observed to form below the Go/G1 region, denoted as the A0 region. The presence of cells in this region was consistent with the presence of internucleosomal DNA fragments as determined by gel electrophoresis. Inhibitors of transcription, translation and endonuclease activity, and a glucocorticoid receptor antagonist prevented accumulation of cells in this region. Irradiation of mouse thymocytes also produced a population in the A0 region. Cells in this region are believed to have undergone glucocorticoid‐induced DNA fragmentation. This method represents a useful alternative to whole cell lysate assays, since apoptosis can be evaluated on an individual cell basis.
Loosening of cell cycle controls of human lymphocytes under the action of tumour promoter TPA
Authors: Vinogradov A. E. , Ezhevsky S. A. , Rosanov J. M. , Kazhdan I. A. , Zweibach A. S.
Published: May 2008

Abstract. The effect of tumour promoter TPA (12‐0‐tetradecanoylphorbol‐13‐acetate) on the cell cycle of human peripheral blood lymphocytes stimulated by phyto‐haemagglutinin (PHA) in vitro was studied and it was found that TPA caused cells to accumulate in all the cell cycle phases. This accumulation took place preferentially at later culture passages, when lymphocytes stimulated by PHA alone stopped mainly in G0/G1 phases. Other effects of TPA were cell induction to enter higher DNA ploidy and to survive and even synthesize DNA under colchicine block of mitosis or under cytochalasin block of cytokinesis. In addition, in experiments in which a transitory block through the G1 phase of cell cycle was applied with use of aminopterin, we could show that a fraction of TPA‐treated cells still entered the active phase of DNA synthesis. These findings suggest that TPA causes cell cycle controls to become loose, thereby enhancing adaptability of human lymphocytes to various hindrances in the course of cell cycle and eventually causing them to acquire characteristics known to be common for tumour cells.
The type, origin and function of the odontogenic cells of continuously growing guinea‐pig molars
Authors: Nataatmadja M. I. , Orams H. J. , Reade P. C.
Published: May 2008

Abstract. Little is known about which cell types act as the origin of odontogenic stem cells and how these stem cells differentiate to become functional. In the present study 3‐day‐old guinea‐pigs were injected with tritiated thymidine, killed at intervals and their molars studied. The odontogenic cells were found to originate from the stem cells which by autoradiography were shown to be slow cycling and lightly labelled. As they differentiated they became heavily labelled and were designated transit ‘dividing cells’. With further differentiation the cells were unlabelled and were designated ‘simple transit cells’.

It was concluded from the present study that outer enamel epithelium functioned as the source of all odontogenic epithelium; that primitive mesenchyme around the cervical loop functioned as the source of all odontogenic mesenchyme and that all of the transit cells which differentiated to become functional migrated coronally.
The chronological relationship between the thickening of smooth muscle, epithelial cell proliferation and myenteric neural denervation in the rat jejunum
Authors: Zucoloto S. , Silva J. C. , Oliveira J. S. M. , Muccillo G.
Published: May 2008

Abstract. The jejunum of rats was treated by serosal application of a 0.2% solution of benzalkonium chloride (BAC) for 30 min. Control animals were treated with saline (0.9% NaC1). The animals were allocated to eight groups of 10 rats each and sacrificed 15, 30, 45, 60 days after BAC treatment. Segments were removed from the jejunum for neuronal counting, measurement of the smooth muscle area and morphokinetic study of the epithelium. There was a significant reduction in neuron number in the myenteric plexus 30 days after BAC treatment, thickening of smooth muscle 15–60 days after BAC treatment, but no change in epithelial cell proliferation in the jejunum at either time.
Protein synthesis inhibitors, like growth factors, may render resting 3T3 cells competent for DNA synthesis: a radioautographic and cell fusion study
Authors: Setkov N. A. , Kazakov V. N. , Rosenwald I. B. , Makarova G. F. , Epifanova O. I.
Published: May 2008

Abstract. Serum‐deprived (0.1–0.2%) resting NIH 3T3 mouse fibroblasts pre‐incu‐bated with cycloheximide (7.5 μ/ml), or puromycin (10 μ/ml), were fused with stimulated cells taken 10 h after changing the medium to one containing 10% serum, and DNA synthesis was investigated in the nuclei of monokaryons, homodikaryons and heterodikaryons using radioautography with the double‐labelling technique. Pre‐incubation of resting cells with inhibitors of protein synthesis for 1–4 h abolished their ability to suppress DNA synthesis in stimulated nuclei in heterokaryons. Three hours after the removal of cycloheximide from the medium, the resting cells acquired once again the inhibitory capacity for entry of stimulated nuclei into the S period. This inhibitory influence disappeared also in the case of post‐fusion cycloheximide application as well as following an 8–12 h pre‐treatment of resting cells with actinomycin D (1 μ/ml) prior to fusion. Pre‐incubation of resting cells for 12 h with PDGF (1 u/ml‐) followed by an 8–48 h incubation in serum‐free medium stimulated the onset of DNA synthesis. A brief exposure (45 min) of resting cells to cycloheximide (7.5 μ/ml), or puromycin (7.5 μ/ml), exerted a similar effect, inducing by itself the entry of cells into the S period. The results support the assumption that acquirement, by resting cells, of competence for DNA replication includes as a necessary step the down‐regulation of intracellular growth inhibitors whose formation depends on protein synthesis.
Spermatogonial apoptosis has three morphologically recognizable phases and shows no circadian rhythm during normal spermatogenesis in the rat
Authors: Allan D. J. , Harmon B. V. , Roberts S. A.
Published: May 2008

Abstract. In this study we examined the possibility that regular or circadian fluctuations occur in the frequency of spontaneous spermatogonial apoptosis. Apoptosis of A2, A3 and A4 type spermatogonia occurring spontaneously in the normal rat testis was studied by light and electron microscopy. Normal and apoptotic A3 spermatogonia were quantified in 36 animals killed at two‐hourly intervals over a 24 h period. Three sequential phases of spermatogonial apoptosis were defined and quantified separately: (i) an early phase in which cells showed margination of nuclear chromatin, (ii) an intermediate phase in which phagocytosed apoptotic bodies were partly degraded and (iii) a late phase in which only debris of degraded apoptotic bodies was evident. Groups of spermatogonia linked by intercellular bridges underwent apoptosis synchronously. Normal and apoptotic A3 spermatogonia occurred at a mean frequency of 33.4 and 9.6 per 10 seminiferous tubule profiles respectively; there was a large variation in these frequencies between animals, but no peaks or circadian periodicity were detected. Progressive degradation of apoptotic bodies was evident, the average ratios of intermediate and late bodies to early bodies being 1.5 and 3.5, respectively. Absence of a circadian rhythm in these data does not exclude the possibility that initiation of apoptosis in susceptible spermatogonial clones is synchronous, and that affected clones undergo lag periods of differing duration before expressing morphological apoptosis.
G1shortening following unbalanced growth a specific v. nonspecific effect
Authors: Goswami P. C. , Higashikubo R. , Tolmach L. J. , Roti J. L. Roti
Published: May 2008

Abstract. The synthesis and abundance of proteins were examined in synchronous populations of HeLa cells under conditions in which the lengthening of S phase, by inhibiting DNA synthesis, resulted in shortening of G1 in the subsequent generation. Mitotically collected cells were resynchronized by incubation with 3 PM aphidicolin from 3 to 12 h after mitotic selection; they were blocked again at various times thereafter to induce unbalanced growth. Cells were labelled with [35S]‐methionine before and after release from the block to study the changes in protein synthesis. Triton X‐100 soluble and insoluble proteins were analysed by 7–15% gradient SDS‐PAGE, and radioactivity incorporation was quantified by liquid‐scintillation counting. The degree of G1 shortening correlated with S phase position, increasing gradually from early S and reaching maximum when cells were blocked half‐way through S phase. Synthesis of proteins of 120, 66, and 51 kDa was stimulated, and synthesis of a new protein of 57kDa was observed, in cells in which DNA synthesis had been blocked in mid‐S. These proteins also showed increased accumulation. These results suggest that the shortening of GI, induced by the prior arrest of cell‐cycle progression, is associated with synthesis of specific proteins rather than the non‐specific accumulation of cellular proteins through unbalanced growth.
Cell death induced by vincristine in the intestinal crypts of mice and in a human Burkitt's lymphoma cell line
Authors: Harmon B. V. , Takano Y. S. , Winterford C. M. , Potten C. S.
Published: May 2008

Abstract. Although vincristine is widely used clinically in the treatment of some human cancers, its mechanism of action has not been clearly established. In this study, the patterns of cell death induced y vincristine in the intestinal crypts of mice and in a human Burkitt's lymphoma cell line were investigated by light and electron microscopy. Vincristine was found to enhance apoptosis of interphase cells in both systems and also to cause the arrest of cells in mitosis, the latter effect being ore pronounced in the intestinal crypts. Arrested mitotic cells went on to die by a process that had a number of features in common with apoptosis. These include compaction of chromatin (following coalescence of chromosomes), condensation of the cytoplasm, initial preservation of organelle integrity, and eventually the fragmentation of the cell into a number of membrane‐enclosed bodies which are morphologically similar to conventional apoptotic bodies. The results suggest that the cytocidal effect of vincristine is not solely dependent on metaphase arrest but is a cumulative one, resulting both from apoptosis of interphase cells and the ‘apoptotic‐like’ death of cells arrested in metaphase.
The mechanism of action of the tetrapeptide Acetyl‐N‐Ser‐Asp‐Lys‐Pro (AcSDKP) in the control of haematopoietic stem cell proliferation
Authors: Robinson S. , Lenfant M. , Wdzieczak‐Bakala J. , Melville J. , Riches A.
Published: May 2008

Abstract. The mechanism of action of the haemoregulatory tetrapeptide Acetyl‐N‐Ser‐Asp‐Lys‐Pro (AcSDKP, Mr = 487 amu), was investigated using an in vitro assay of a murine high proliferative potential colony‐forming cell (HPP‐CFC) which responds to proliferation regulators of the haematopoietic stem cell population. AcSDKP had no direct inhibitory effect on the number, or the proportion in S phase, of the committed granulocyte‐macrophage progenitor (GM‐CFC), or cycling HPP‐CFC populations. However, AcSDKP blocked the action of a stimulator of haematopoietic stem cell proliferation, preventing the switching of quiescent HPP‐CFC into cell cycle. It would appear that AcSDKP exerts its inhibitory haemoregulatory role indirectly, by preventing stimulator action on haematopoietic stem cells.
Effects of growth media on cell cycle progression in CHO cells exposed to the radioprotector WR‐1065
Authors: Murley J. S. , Grdina D. J. , Meechan P. J.
Published: May 2008

Abstract. WR‐1065 (2‐[(aminopropyl)amino]ethanethiol) reduces cytotoxic and mutagenic effects caused by exposure of cells to radiation and chemotherapeutic drugs, but the mechanisms involved are not fully known. We have observed an accumulation of cells in G, in WR‐1065 treated Chinese hamster ovary cells grown in a‐minimal essential medium, while others have found no cell cycle effects in WR‐1065 treated Chinese hamster ovary cells grown in McCoy's 5A medium. To determine if the two types of media had an effect on cells treated with WR‐1065, we examined survival and cell cycle progression. Population doubling times of 12 h were observed for cells grown in both media. Incubation of AA8 cells grown in McCoy's 5A medium with 4 mM WR‐1065 30 min prior to and during irradiation with 13′Cs gamma‐rays resulted in a protection factor of 2.2, in close agreement with the value of 2.0 we previously obtained for AA8 cells grown in α‐minimal essential medium. Treatment with WR‐1065 caused an alteration in the cell cycles of cells grown in both media. An increase in the G2 population and a decrease in the G1 population was observed in cells incubated up to 3 h in the presence of 4 mM WR‐1065, with a redistribution of the cells throughout the cell cycle occurring following removal of the drug. These data suggest that exposure of cells to WR‐1065 is the cause of perturbations in cell cycle progression, and is not affected by the type of medium the cells are grown in.
Simultaneous assessment of migration and proliferation of murine fibrosarcoma cells, as affected by hydroxyurea, vinblastine, cytochalasin B, Razoxane and interferon
Authors: Haug I. J. , Siebke E. M. , Grimstad I. A. , Benestad H. B.
Published: May 2008

Abstract. Using porous cell culture chambers, we have simultaneously assessed growth and locomotion of cancer cells to investigate whether certain agents affect cell motility in addition to cell division. First, cells from a murine fibrosarcoma cell line, 1.0/L1, were grown in ordinary flask cultures to determine appropriate cell inocula. Doses of agents were selected to reduce the final 4 day culture cellularity to about 50%, when present during the last two days of culturing. Secondly, the effects of these agents on cell numbers in the porous chambers and on cell migration out of the chambers (‘emigration fraction’) were recorded. We also examined, using a similar type of porous chamber, whether the agents could affect leucocyte chemotaxis. Hydroxyurea (an inhibitor of DNA synthesis) reduced cancer cell emigration as well as cell growth, without interfering with leucocyte chemotaxis. Cytochalasin B (a microfilament disrupting agent) inhibited cancer cell motility and growth, as well as leucocyte chemotaxis. Vinblastine (a microtubule disrupting agent), at the very low dose chosen, reduced cancer cell growth, but did not consistently affect the migration of either cell type. The experimental anti‐metastasis agent Razoxane reduced growth, but had no detectable effects on motility. High doses of natural murine interferon–α/β weakly inhibited both cancer cell growth and locomotion. This motivates for further studies of these and other cytokines, as treatment with agents inhibiting cancer cell locomotion might possibly prevent peri‐operative spread of cancer in patients.
Proliferation kinetics and PCNA expression of HL‐60 cells following ionizing irradiation and granulocytic differentiation
Authors: Sendler A. , Kaffenberger W. , Nuyken I. , Van Beuningen D.
Published: May 2008

The human promyelocytic leukaemia cell line, HL‐60, was investigated with regard to proliferation and terminal differentiation following irradiation. The cells were X‐irradiated and induced with 1.25% dimethyl sulfoxide (DMSO) towards the granulocytic lineage. Proliferation was measured via cell growth, clonogenicity and the bromodeoxyuridine/DNA incorporation assay. Immunohistochemical detection of proliferating cell nuclear antigen (PCNA) expression was used to discriminate cycling from non‐cycling cells. The differentiation obtained was proved by testing for the immune function of the respiratory burst (NBT reduction test). The HL‐60 cells studied revealed a high radiosensitivity (D0= 0.63 Gy). After induction with DMSO, declines in cell growth, clonogenicity and PCNA positivity of the cells indicated a decrease in proliferation and an increase in differentiation. Starting on day 2 in culture, irradiation after seeding with 1 Gy accelerated the loss of the PCNA expression in induced cells (46%v. 3% PCNA‐negative control cells on day 3). Induced cells gained the capability of exerting the respiratory burst, which was found to be dose‐dependent radiosensitive (42% and 12% NBT‐positive cells after 1 and 2 Gy, respectively, v. 53% NBT‐positive control cells on day 8). Subpopulations in the cell line were evident in all parameters investigated. We discuss the HL‐60 cell, not only as a model comparable to human progenitor cells, but also as a suitable tool in radiobiological research with regard to proliferation and differentiation following ionizing irradiation.
Transforming growth factor‐α immunoreactivity in a variety of epithelial tissues
Authors: Alison* M. R. , Nasim M. M. , Anilkumar T. V. , Sarraf C. E.
Published: May 2008

Abstract. The immunohistochemical expression of transforming growth factor‐alpha (TGFα) has been examined in a range of normal adult epithelial tissues from both man and rat using an anti‐hTGFα monoclonal antibody (GF10). No differences in distribution were apparent between man and rat. In the continually renewing epithelium of the gastrointestinal tract, no staining was seen within the proliferative compartments, but strong immunoexpression was noted in various differentiated populations. In the testis, the spermatogonia were unstained, but the more luminally orientated germ cells were strongly positive. In the gastrointestinal tract, at least, any mitogenic action of TGFα must be mediated through a relatively long paracrine loop. In contrast, the differentiated parenchyma of kidney, salivary gland and liver remained unstained apart from collecting ducts in the kidney, striated ducts in salivary glands and bile ducts in the liver. The association of TGFα with tubule formation was reinforced by the very strong staining of newly forming bile ducts in a model of liver oval cell proliferation. Thus, in all the epithelia studied there was a distinct spatial pattern of TGFα immunoreactivity.
A simplified HPLC method for simultaneously quantifying ribonucleotides and deoxyribonucleotides in cell extracts or frozen tissues
Authors: Cross D. R. , Miller B. J. , James S. J.
Published: May 2008

Agents and conditions that induce alterations in deoxyribonucleotide pools can have important regulatory effects on the rate of DNA synthesis as well as cell cycle progression. A simplified procedure for the separation of both ribonucleoside triphos‐phates (NTP) and deoxyribonucleoside triphosphates (dNTP) is presented which utilizes reversed phase high‐performance liquid chromatography coupled with diode array detection. The simultaneous resolution of NTP and dNTP peaks within the same cell extract effectively eliminates the need for post‐extraction steps such as periodate oxidation and/or boronate affinity chromatography previously used to degrade or isolate co‐eluting NTP from dNTP. The resolution of two nucleotides, dGTP and ADP, was found empirically to vary with the efficiency of the C18 column. High efficiency columns (>90 000 plates/m) provided good separation; however, less efficient columns resulted in co‐elution of dGTP and ADP. These co‐eluting nucleotides can be accurately quantified, if necessary, using diode array technology and a mathematical expression which incorporates molar peak coefficients and peak areas obtained by monitoring at dual wavelengths. Tissue samples or single cell suspensions were extracted with trichloroacetic acid and the neutralized extract was injected directly into the column without prior lyophilization. The per cent recovery of standards was .99% and replicate extractions within or between samples were highly reproducible (sd<5%). The single step method described minimizes potential losses associated with post‐extraction manipulation and provides the capability to examine alterations in nucleotide precursor–product metabolism under various physiological and pharmacological conditions.
A comparison of different methods to determine cell proliferation by flow cytometry
Authors: Zölzer F. , Streffer C. , Pelzer T.
Published: May 2008

Abstract. The proliferation of human melanoma cells (MeWo) in vitro was studied with a number of different techniques. In particular, we compared the expression of PCNA and the Ki‐67 antigen on the one hand with BrdU pulse and continuous labelling on the other. Two‐dimensional flow cytometry (with DNA content as a second parameter) was employed to discriminate between cycling and non‐cycling cells as well as cells in the G1, S and G2 phases of the cycle. Cell cultures in different stages of growth were analyzed. We found that the percentage of anti‐PCNA and Ki‐67 positive cells agreed very well with the BrdU pulse and continuous labelling index, respectively. Our data further support the assumption that under certain conditions PCNA is a marker of S‐phase cells, whereas Ki‐67 can be used to quantify the growth fraction. Possible pitfalls of the techniques are discussed.
A comparison of time‐lapse cinemicrography and flow cytometry for the study of accelerated cell‐cycle transit
Authors: Higashikubo R. , Goswami P. C. , Roti J. L. Roti
Published: May 2008

Abstract. Two methods for the study of cell‐cycle progression, time‐lapse cinemicrography (TLCM) and flow cytometry (FCM), were compared for their ability to measure the shortening of cell‐cycle transit time induced by temporary inhibition of DNA synthesis. DNA synthesis was reversibly inhibited by aphidicolin (APH) in synchronized HeLa cells obtained by mitotic collection. TLCM directly measured intermitotic time intervals and thereby directly obtained the cell‐cycle transit time distribution. In contrast, FCM measured time dependent changes in the fractions of cells in the cell‐cycle phases from which the distribution of cells traversing a cell‐cycle boundary, such as that between G1 and S phase, was determined. Nevertheless, both methods provided equivalent measures of the cell‐cycle transit time and its dispersion. However, TLCM apeared to provide a better measure of skewness of the transit time distribution than did FCM. Further, both methods were able to detect changes in the cell cycle transit on the order of 1 h or less. The TLCM data showed a greater precision (due to a larger number of data points) than that from FCM. However, FCM was able to directly measure changes in the transit of G1 phase whereas TLCM would require two different experiments to make a similar determination. The results obtained in this study show that FCM can replace TLCM to study most aspects of cell‐cycle progression.
Expression of myc, fos and Ha‐ras associated with chemically induced cell proliferation in the rat liver
Authors: Goldsworthy T. L. , Goldsworthy S. M. , Sprankle C. S. , Butterworth B. E.
Published: May 2008

Abstract. Events secondary to induced cell proliferation may play a role in the carcinogenic process. These studies investigated the expression of genes associated with growth control in response to two types of cell proliferation stimuli in the livers of male F344 rats. Regenerative hepatocyte proliferation after partial hepatectomy or a single dose of carbon tetrachloride, and mitogenic liver hyperplasia induced by a single dose of pheno‐barbital or WY‐14,643 were assessed by thymidine incorporation and quantitative autoradiography. The expression of myc, fos, and Ha‐ras was evaluated by Northern blot analysis of liver derived poly(A)+ mRNA from these same animals. After each treatment, the level of hepatocyte proliferation (labelling index 4–32%) was observed to peak between 24 and 48 h and return to control values by 8 days. In every case, a peak in myc expression was seen between 0.5 and 18 h depending on the proliferative stimulus treatment. A large peak infos expression was seen at 0.5–2 h but only with the cytotoxic and regenerative proliferative treatments partial hepatectomy or carbon tetrachloride. A broad peak in Ha‐ras expression was observed 12 to 36 h after each treatment. These data demonstrate transient expression of these genes following the synchronous induction of hepatocyte proliferation. The increased expression offos upon treatment with cytotoxicants, but not mitogens, suggests different modes of growth regulation that may be important in understanding the induction of cell proliferation by these two types of agents.
The effect of folic acid and/or methionine deficiency on deoxyribonucleotide pools and cell cycle distribution in mitogen‐stimulated rat lymphocytes
Authors: James S. J. , Miller B. J. , McGarrity L. J. , Morris S. M.
Published: May 2008

Abstract. Folate deficiency will induce abnormal deoxynucleoside triphosphate (dNTP) metabolism because folate‐derived one‐carbon groups are essential for de novo synthesis of purines and the pyrimidine, thymidylate. Under conditions of methionine deprivation, a functional folate deficiency for deoxynucleoside triphosphate synthesis is induced as a result of the irreversible diversion of available folates toward endogenous methionine resynthesis from homocysteine. The purpose of the present study was to examine the effect of nutritional folate and/or methionine deprivation in vitro on intracellular dNTP pools as related to DNA synthesis activity and cell cycle progression. Primary cultures of mitogen‐stimulated rat splenic T‐cells were incubated in complete RPMI 1640 medium or in custom‐prepared RPMI 1640 medium lacking in folic acid and/or methionine. Parallel cultures, initiated from the same cell suspension, were analysed for deoxyribonucleotide pool levels and for cell proliferation. The distribution of cells within the cell cycle was quantified by dual parameter flow cytometric bromodeoxyuridine/propidium iodide DNA analysis which allows more accurate definition of DNA synthesizing S‐phase cells than the traditional DNA‐specific staining with propidium iodide alone. Relative to cells cultured in complete RPMI 1640 media, the cells cultured in media deficient in folate, methionine or in both nutrients manifested increases in the deoxythymidylate pool and an apparent depletion of the deoxyguanosine triphosphate pool. Both adenosine triphosphate and nicotinamide adenine diphosphate levels were significantly reduced with single or combined deficiencies of folate and methionine. These nucleotide pool alterations were associated with a decrease in the proportion of cells actively synthesizing DNA and an increase in cells in G2+ M phase of the cell cycle. Folate deprivation in the presence of adequate methionine produced a moderate decrease in DNA synthesizing cells over the 68 h incubation. However, methionine deprivation, in the presence or absence of folate, severely compromised DNA synthesis activity. These results are consistent with the established ‘methyl trap’ diversion of available folates towards the resynthesis of methionine from homocysteine and away from nucleotide synthesis. The data confirm the metabolic interdependence of folic acid and methionine and emphasize the pivotal role of methionine on the availability of folate one‐carbon groups for deoxynucleotide synthesis. The decrease in DNA synthesis activity under nutrient conditions that negatively affect nucleotide biosynthesis suggest a possible role for abnormal dNTP metabolism in the regulation of cell cycle progression and DNA synthesis.
Expression of the hepatocyte growth factor and c‐MET genes during furan‐induced regenerative cell proliferation in the livers of B6C3F1 mice and F‐344 rats
Authors: Sprankle C. S. , Goldsworthy T. L. , Goldsworthy S. M. , Wilson D. M. , Butterworth B. E.
Published: May 2008

Abstract. Chronic administration of furan has been reported to produce hepatocellular carcinomas in male and female mice and male rats and cholangiocarcinomas in male and female rats. The weight of evidence indicates that furan is not genotoxic and tumours appear to arise secondary to initiation and promotional events associated with furan‐induced necrosis and regenerative cell proliferation. Identification of altered patterns of gene expression related to these events may provide insight into some of the underlying factors involved in this carcinogenic process. Hepatocyte growth factor (HGF) and its receptor, the protein product of the c‐met oncogene, play an important role in regenerative growth of liver. These experiments examined the relationship between induced cell proliferation and expression of the HGF and c‐met genes in liver after treatment with furan. Male rats were administered a single necrosis‐inducing dose of furan, and male mice and male and female rats were treated daily with furan by gavage for up to six weeks under conditions of the cancer bioassay. Northern blot analysis of mRNA isolated from livers of those rats administered a single dose of furan detected a transient increase in expression in the HGF gene associated with the resulting wave of regenerative cell proliferation, but no increase in the expression of c‐met. No increase in expression of either gene was detected in liver tissue from any of the animals treated for up to six weeks, even though elevated rates of regenerative hepatocyte proliferation were sustained. Thus, given the detection limits of the techniques used here, sustained increases in expression of these genes are not required to maintain the regenerative response.
Cell proliferation kinetics of normal and tumour tissue in vitro: quiescent reproductive cells and the cycling reproductive fraction
Authors: Baker F. L. , Sanger L. J. , Rodgers R. W. , Jabboury K. , Mangini O. R.
Published: May 2008

Current methods for measuring the cell kinetics of human tumours are made and interpreted within the context of a simplistic two compartment model for cell proliferation, consisting of cells that are cycling and those that are not. It is now recognized that the non‐cycling compartment of many tumours is heterogeneous, composed of non‐reproductive end‐stage cells and reproductive cells that are dormant/quiescent. We have developed an in vitro analysis that distinguishes for the first time quiescent reproductive cells from non‐reproductive end‐stage cells and have integrated this analysis with monolayer clonogenic and suicide assays to simultaneously quantitate the duration of the cell cycle and reproductive cells that are: cycling, quiescent, clonogenic, and non‐reproductive end‐stage cells. We have defined a new parameter, the Cycling Reproductive Fraction (CRF), which is the cycling cell population referenced specifically to the reproductive cell population. Measurements of CRF from 72 tumour biopsies and from 5 normal foreskins showed that CRF approached 100% in some tumours; however, CRF showed near normal values (< 1%) in others suggesting that cell cycle control may be maintained in some tumours. Because of CRF's improved specificity, we believe that CRF may enhance classification, prognostication, and the optimization and prediction of response to chemotherapy.
Existence of a commitment program for mitosis in early G1 in tumour cells
Authors: Larsson O. , Zetterberg A.
Published: May 2008

The proliferation of normal non‐tumourigenic mouse fibroblasts is stringently controlled by regulatory mechanisms located in the postmitotic stage of G1 (which we have designated G1 pm). Upon exposure to growth factor depletion or a lowered de novo protein synthesis, the normal cells leave the cell cycle from G1 pm and enter G0. The G1 pm phase is characterized by a remarkably constant length (the duration of which is 3 h in Swiss 3T3 cells), whereas the intercellular variability of intermitotic time is mainly ascribable to late G1 or pre S phase (G1 ps) (Zetterberg & Larsson (1985) Proc. Natl. Acad. Sci. USA82, 5365). As shown in the present study two tumour‐transformed derivatives of mouse fibroblasts, i.e. BPA31 and SVA31, did not respond at all, or only responded partially, respectively, to serum depletion and inhibition of protein synthesis. If the tumour cells instead were subjected to 25‐hydroxycholesterol (an inhibitor of 3‐hydroxy‐3 methyglutaryl coenzyme A reductase activity), their growth was blocked as measured by growth curves and [3H]‐thymidine uptake. Time‐lapse analysis revealed that the cells were blocked specifically in early G1 (3‐4h after mitosis), and DNA cytometry confirmed that the arrested cells contained a G1 amount of DNA. Closer kinetic analysis revealed that the duration of the postmitotic phase containing cells responsive to 25‐hydroxycholesterol was constant. These data suggest that transformed 3T3 cells also contain a ‘G1 pm program’, which has to be completed before commitment to mitosis. By repeating the experiments on a large number of tumour‐transformed cells, including human carcinoma cells and glioma cells, it was demonstrated that all of them possessed a G1 pm‐like stage. Our conclusion is that G1 pm is a general phenomenon in mammalian cells, independent of whether the cells are normal or neoplastic.
Effects of omeprazole on cell kinetics of carcinogen‐induced colon tumours in rats
Authors: Hurwitz A. , Sztern M. I. , Looney G. A. , Pinson D. M. , Bauer K. D. , Kimler B. F.
Published: May 2008

This study was designed to evaluate the effects of hypergastrinaemia induced via suppression of gastric acid by omeprazole on carcinogen‐induced colon cancer in rats. The carcinogen methylazoxymethanol (MAM), 30 mg/kg, was administered intraperitoneally at 6‐weekly intervals to Sprague‐Dawley rats. Four weeks after the last MAM injection, the first daily dose of omeprazole, 40 mg/kg, was given by gastric gavage to one group of rats, and the rest were given buffered methylcellulose vehicle. After 10 weeks of daily omeprazole or vehicle, the rats were anaesthetized with ether, blood samples obtained, and animals sacrificed. Gastrin levels in serum from omeprazole‐treated rats were elevated nearly six‐fold. DNA and RNA levels in gastric mucosa were unchanged by omeprazole, but protein content was somewhat reduced. No biochemical or histological changes related to omeprazole treatment were observed in normal colon. The number of tumours, tumour volumes, and total tumour burden were not significantly different in colons of vehicle‐ or omeprazole‐treated rats. Analysis by flow cytometry revealed that the S phase fraction was lower in tumour cells from omeprazole‐treated animals; and that the frequency of DNA aneuploidy was also reduced. The results indicate that while omeprazole‐induced suppression of stomach acid in rats elevates levels of gastrin in serum, it does not substantially alter the biochemical or cellular characteristics of carcinogen‐induced colon tumours.
Proliferation of mature and immature subpopulations of bronchoalveolar monocytes/macrophages and peripheral blood monocytes
Authors: Van Hal P. Th. W. , Wijkhuijs J. M. , Mulder P. G. H. , Hoogsteden H. C.
Published: May 2008

A continuous influx of peripheral blood monocytes (PBM) to the lung is thought to maintain the local population of alveolar macrophages (AM). However, local proliferation of a small subpopulation of AM has been demonstrated in animal studies and in humans. AM exhibit a great heterogeneity with regard to their morphology (cell size, shape of nucleus), immunophenotype (expression of CD14 and RFD9 antigen), and function. Part of this heterogeneity may be explained by the presence of different maturation stages of AM, ranging from small immature, CD14+ RFD9− PBM‐like cells to large, CD14− RFD9+ mature AM. These findings prompted us to study whether proliferation of PBM and AM is related to their stage of maturation.

The expression of the proliferation marker Ki‐67 was studied in AM from both healthy volunteers and patients suffering from sarcoidosis. Using double immunofluorescence staining, we studied proliferation of immature, CD14+ AM, and mature, RFD9+ AM in sarcoidosis, and we compared this with PBM. A significantly larger percentage of AM in general expressed Ki‐67 antigen in sarcoidosis (3.0 (median); range 1.1–5.5) as compared with healthy volunteers (0.8; 0.2‐1.3). In sarcoidosis, proliferation was observed in both the immature and the mature subpopulation of AM. Proliferating PBM were rarely observed [less than 0.2% of the CD14+ mononuclear cells (MNC)] both in healthy volunteers and sarcoidosis patients. A small subpopulation of PBM showed a weak expression of RFD9 antigen (less than 1% of MNC). Interestingly, proliferation of PBM was concentrated in this subpopulation (15% of the RFD9+ MNC).

These data show that even mature AM, which are generally thought to be terminally differentiated cells with little capacity to replicate, are able to proliferate, whereas a relatively very low percentage of their precursors in the blood circulation proliferates. Furthermore, the findings suggest that lung tissue in sarcoidosis creates an environment which promotes proliferation of monocytic cells.
Purine suppression of proliferation of Sertoli‐like TM4 cells in culture
Authors: Shaban M. , Smith R. A. , Stone T. W.
Published: May 2008

The effect of adenosine and related compounds on the proliferation of cultured TM4 cells, a Sertoli‐like cell line, has been examined. Adenosine, as well as A1 and A2 adenosine receptor agonists (cyclohexyladenosine and N6‐[2‐(3,5‐dimethoxyphenyl)‐2‐(2‐methylphenyl)ethyl]adenosine) inhibited cell proliferation. These effects were prevented by 8‐cyclopentyl theophylline, 1,3‐dimethyl‐propargylxanthine and caffeine, antagonists at the A1, A2 and both receptors, respectively. The xanthines had no effect by themselves and, consistent with this, the bathing medium was found not to contain detectable levels of adenosine. It is concluded that TM4 cell proliferation can be regulated by receptors for adenosine.
Cell cycle analysis by the relative movement approach: effect of variability across S‐phase of DNA synthesis rate
Authors: Bertuzzi A. , Grosso N. Del , Gandolfi A. , Sinisgalli C. , Starace G.
Published: May 2008

Cell populations pulse‐labelled with BrdUrd, and sampled at increasing times after the pulse, yield DNA‐BrdUrd distributions from which the relative movement (RM) and the depletion function (DF) of labelled, undivided cells can be calculated. In this paper we present an extension of the equation for the time course of RM, given by White and Meistrich (Cytometry 1986, 7, 486–490), to the case in which the rate of DNA synthesis changes across S‐phase. Some modalities of cell loss were also considered. Computer simulations showed that different patterns of DNA synthesis rate across S‐phase can result in appreciably different RM curves. An analytical expression of the RM curve, in which the variability across S‐phase of the rate of DNA synthesis is accounted for by only one parameter, was proposed. This expression was used for the simultaneous fitting of time sequences of RM and DF data of U937 cells, in order to estimate the phase transit times TS and TG2+M, and the potential doubling time Tpot. The use of the extended model gave better results than those obtained under the assumption of constant rate of DNA synthesis across S‐phase.
The effect of extracellular calcium on colonocytes: evidence for differential responsiveness based upon degree of cell differentiation
Authors: Buras R. R. , Shabahang M. , Davoodi F. , Schumaker L. M. , Cullen K. J. , Byers S. , Nauta R. J. , Evans S. R. T.
Published: May 2008

Calcium supplementation decreases the incidence of colon cancer in animal models and may prevent colon cancer in man. Potential mechanisms include binding of mitogens and direct effects of calcium on colonic epithelial cells. In this study, the effects of extracellular calcium on epithelial cell growth and differentiation were studied in three colon carcinoma and two colonic adenoma cell lines. The characteristics studied included morphology, cell cycle kinetics, [Ca2+]IC (intracellular calcium concentration), proliferation, and expression of differentiation markers such as carcinoembryonic antigen (CEA) and alkaline phosphatase (AP). Sodium butyrate (NaB) and 1,25‐dihydroxyvitamin D3 were used as controls in the latter three assays as these two agents are known differentiating agents. Alteration of [Ca+2]EC (extracellular calcium concentration) did not affect carcinoembryonic antigen (CEA) or alkaline phosphatase (AP) expression. NaB enhanced the expression of AP three‐fold and CEA five‐fold. This effect was augmented by increasing [Ca2+]EC. The exposure of cells to 1,25‐(OH)2‐Vitamin D3 increased CEA but not AP. [Ca2+]IC increased in response to 1,25‐(OH)2‐vitamin D3 and NaB but not with variation in [Ca2+]EC. Increased [Ca2+]EC inhibited proliferation of well‐differentiated cells, but had no effect on poorly‐differentiated cells. Morphological studies showed that extracellular calcium was necessary for normal cell—cell interactions.

These studies have demonstrated direct effects of calcium on colonic epithelial cells which may contribute to the protective effects of dietary calcium against colon cancer. Loss of responsivess to the antiprotective effects of [Ca2+]EC with de‐differentiation suggests that calcium supplementation may be most beneficial prior to the development of neoplastic changes in colonic epithelium.
Nucleolin and fibrillarin expression in stimulated lymphocytes and differentiating HL‐60 cells. A flow cytometric assay
Authors: Méhes G. , Pajor L.
Published: May 2008

Nucleolin and fibrillarin are two histone‐like major proteins in the nucleolus that were found to be overexpressed in proliferating cells. Using specific antibodies to either nucleolin or fibrillarin flow cytometric, measurements were carried out to demonstrate quantitative changes of these proteins during lymphocyte mitogenic activation and differentiation of HL‐60 promyelocytic leukaemia cells. The expression of nucleolin increased during lymphocyte stimulation and decreased slowly but constantly in the course of differentiation of HL‐60 cells. Expression of fibrillarin reached a maximum in the first cell cycle and then dropped to a basic level in stimulated lymphocytes. Compared to nucleolin, the level of fibrillarin decreased more rapidly and more extensively in differentiating HL‐60 cells. The data support other observations that nucleolin is a stabile structural protein at the ribosomal genes while fibrillarin may have a more specific functional role in nucleologenesis and ribosome production.
Secretion rates and levels of vascular endothelial growth factor in clone A or HCT‐8 human colon tumour cells as a function of oxygen concentration
Authors: Leith J. T. , Michelson S.
Published: May 2008

Molecular and in situ hybridization studies have shown, in a number of cell types, that under hypoxic conditions, vascular endothelial growth factor (VEGF) mRNA expression is up‐regulated and VEGF protein is concomitantly increased. To establish a quantitative relationship between VEGF protein levels and oxygenation, we exposed exponentially growing clone A or HCT‐8 human colon tumour cells in vitro (22 h at 37°C) to oxygen concentrations from 21% (air mixture) to 0.01%. Protein levels in cells and medium were then assayed using an enzyme‐linked immunoabsorbent assay (ELISA). Intracellular levels of VEGF in clone A or HCT‐8 cells exposed to either air (21% O2) or the 0.01% O2 mixture respectively increased from about 73 to 1270, and 1.5 to 1180 pg/106 cells (about 17‐ and 80‐fold increases). The shapes of the response curves (log of the intracellular VEGF concentrations v. log oxygen concentration) for both cell types were sigmoidal. However, intracellular VEGF levels in HCT‐8 cells were always less than that of clone A cells until levels of about 0.3 to 0.1% O2 were reached. Levels of VEGF in the supernatant were also increased after the 22 h hypoxic exposures. Because cell proliferation and clonogenicity were also measured, it was possible to estimate the secretion rates of VEGF for both cell lines as a function of oxygen percentage. For clone A cells, the secretion rate (pg/106 cells/h) in 21% O2 was 62.5. This rate increased to 428.8 pg/106 cells/h at 0.01% O2, a 7‐fold increase. For HCT‐8 cells, levels in the medium at 21% O2 were too low to be measured by ELISA. However, between 10% and 0.01% O2, secretion rates increased from 5.0 to 376.0 pg/106 cells/h, a 75‐fold increase. Therefore, at very low O2 levels, VEGF secretion rates were similar in the two cell lines. We propose that the different VEGF responses of clone A and HCT‐8 colon tumour cells to hypoxic stress in vitro are related to the in vivo observation that the respective hypoxic percentages of solid neoplasms originating from these cell lines are markedly different (i.e. about 3 versus 80%) at equivalent volumes of 750 mm3.