bluuebunny/crossref_metadata_2025 huggingface dataset:
Turnover and Maturation Kinetics In the Hairless Mouse Epidermis. Continuous [3H]Tdr Labelling and Mathematical Model Analyses
Authors: Thorudt E. , Clausen O. P. F. , Aarnaes E.
Published: May 2008

Abstract. Hairless mice were continuously labelled with 10μCi of tritiated thymidine ([3H]TdR) every 4 h for 8 d, and the proportions of labelled basal and differentiating cells were recorded separately. the mitotic rate was measured by the stathmokinetic method and the cell cycle distributions were measured by flow cytometry of isolated basal cells at intervals during the labelling period. the mitotic rate of the [3H]TdR‐injected animals did not deviate from control values during the first 5 d. Computer simulations of the data based on various mathematical models were made, and three main conclusions were obtained: (1) a large spread in transit times through the G1 phase was found, together with a very narrow distribution in maturation time of differentiating cells; (2) about 20% of the differentiating cells were estimated to leave the basal cell layer directly after mitosis. This is consistent with results obtained from different sets of data; and (3) during continuous labelling more than 90% of the cells are labelled during each passage through the S phase.
Flow Cytometric Analysis of the Effects of High Protein Diet On Isolated Rat Liver Mitochondria
Authors: O'Connor J. E. , Kimler B. F. , Vargas J. L.
Published: May 2008

Abstract. We have investigated changes that occur in mitochondria obtained from the livers of rats that had been maintained on a high protein diet (80% casein instead of 20%) for 6 months. Liver homogenates were separated by centrifugation into a mitochondrial fraction, a nuclear fraction and the supernatant fluid of the nuclear fraction (nuclear wash). Rhodamine‐123 was used to selectively stain mitochondria depending upon their membrane. potential. the stained organelles were processed through a flow cytometer where the fluorescent stains were excited by the 488 nm wavelength of a laser and the resultant fluorescence signals analysed. After 6 months on a high protein diet, mitochondria displayed an increase in the fluorescence associated with rhodamine‐123 uptake in both mitochondrial and nuclear wash fractions, while mitochondrial fluorescence in the nuclear fraction showed a heterogeneous distribution. This was interpreted as an increase in membrane potential in most of the liver mitochondria under these nutritional conditions, with a certain degree of heterogeneity. These functional changes may be correlated with morphological alterations previously reported and show the usefulness of flow cytometry for biochemical analysis of isolated mitochondria.
Haemopoietic Inductive Capacity of Irradiated Stromal Cell Layers In Human Micro Long‐Term Bone Marrow Cultures
Authors: Brühl P. , Mergenthaler H.‐G. , Dörmer P.
Published: May 2008

Abstract. In a micro long‐term bone marrow culture (LTBMC) system the effects of irradiation on confluent stromal cell layers were studied. In order to individually analyse the number of granulocyte‐macrophage colony‐forming cells (GM‐CFC) per LTBMC a miniaturized human GM‐CFC assay was established. the normalized GM‐CFC numbers in the micro‐assay compared well with data by the conventional GM‐CFC assay. Pre‐formed stromal cell layers were irradiated with doses up to 20 Gy and subsequently recharged with allogeneic bone marrow cells (BMC). Immediately before recharge the BMC were stromal cell‐depleted by nylon wool filtration. When stromal cell‐depleted BMC were inoculated on empty culture dishes, in vitro haemopoiesis rapidly declined. Sustained GM‐CFC production, however, was seen when these cells were used as a second inoculum. It is concluded that irradiation doses of up to 20 Gy do not cause alteration of the haemopoietic inductive capacity of confluent stromal cell layers.
The Spleen Colony Technique. Ii. Errors In Estimation of the Fraction of Cfu‐S Synthesizing Dna
Authors: Znojil V. , Necas E.
Published: May 2008

A refinement of the method for determining the fraction of CFU‐s in the S phase is presented. the new procedures were tested by means of a model experiment in which the fraction of CFU‐s ‘killed’ was simulated by diluting the cell suspension. Another test was to determine the fraction of the CFU‐s in the S phase using duplicate samples.
The highest labelled cell in the intestinal crypt colum
Authors: Appleton D. R. , Sunter J. P. , Watson A. J.
Published: May 2008

Abstract A formula is given, and proved, for the expected position of the highest labelled cell in an individual crypt column, if the labelling index distribution is known.
Sequential flow cytometric analysis of cell‐cycle related changes in LFA‐1 (CD18/CD11a) expression by Trisomy 21 (Down's syndrome) lymphoblastoid cells*
Authors: McMurray B. P. , Taylor G. M. , Williams A. , D'Souza S. , Harris R.
Published: May 2008

Abstract Lymphocyte function associated antigen 1 (LFA‐1) is a heterodimeric leucocyte adhesion molecule comprising non‐covalently associated 95 kD, CD18 and 180 kD, CD11a subunits. Lymphoblastoid cell‐lines (LCL) derived from persons with Down's syndrome (Trisomy 21) exhibit increased expression of LFA‐1 compared with normal LCL. Although this is probably due to a gene‐dosage related increase in the synthesis of CD18, cell‐cycle differences between Trisomy 21 (T21) and normal LCL could also influence LFA‐1 expression. We have therefore analysed expression of CD18 on G1 and G2M cells using sequential flow cytometry. T21 and normal LCL were co‐stained with the DNA‐binding vital dye HO342 (Hoechst 33342), and with a CD18 monoclonal antibody. The LCL were first sorted on the basis of HO342 staining into G1 and G2M populations, and these fractions then analysed for CD18 expression. Irrespective of the stage of the cell‐cycle, expression of CD18 was increased on T21 compared with normal LCL. Although more CD18 was detected on both T21 and normal G2M compared with G1 cells, the relative density of CD18 in G2M was less than in G1 because G2M cells were larger.
Cell proliferation and polyamine metabolism in 9L cells treated with (2R,5R)‐6‐heptyne‐2,5‐diamine or α‐difluoromethylornithine
Authors: Milam K. M. , Deen D. F. , Marton L. J.
Published: May 2008

Abstract We studied the effects of the ornithine decarboxylase inhibitors (2R,5R)‐6‐heptyne‐2,5‐diamine (R,R,‐MAP) and α‐difluoromethylornithine (DFMO) on cell proliferation and polyamine metabolism in 9L rat brain tumour cells. Treatment with 5 μM R,R‐MAP inhibited cell proliferation to the same extent as did treatment with 1 mM DFMO. Both inhibitors depleted putrescine and spermidine concentrations to less than detectable levels within 24 h and 48 h of drug treatment, respectively; spermine levels were not affected significantly by either inhibitor. The effects of DFMO on 9L cell cycle kinetics were similar to those of R,R‐MAP. During the first 3 days of treatment, both drugs caused an accumulation of cells in G1 and a reduction of cells in S phase, as compared with control cells with a slowing in the rate of cell cycle traverse. In cultures seeded at low (1 × 105), medium (5 × 105), or high (2 × 106) cell densities in a 25 cm2 flask, inhibition of cell proliferation and polyamine depletion by both R,R‐MAP and DFMO was more pronounced at the lower densities relative to the density‐matched control cells. Thus, R,R‐MAP was a more potent inhibitor of ornithine decarboxylase than was DFMO in 9L cells, and the inhibitory effects of both compounds on cell proliferation and polyamine biosynthesis were greater in actively proliferating cells.
Effect of the duration of infusion of urogastrone* on intestinal regeneration in rabbits
Authors: Thompson J. S. , Saxena S. K. , Sharp J. G.
Published: May 2008

Abstract Urogastrone (UG) increases the rate of mucosal regeneration on patched intestinal defects. Our aim was to determine the effect of the duration of UG administration on regeneration of serosa patched ileal defects in rabbits. Group I (n= 18) were controls. Group II (n= 15), Group III (n= 10) and Group IV (n= 5) received UG 1.5 μ/kg/h subcutaneously for 7 days, 14 days or 21 days respectively. Animals were sacrificed at 7 day intervals up to 21 days after patching. Neomucosal growth was significantly greater in the animals receiving UG and was greatest in Group IV. Group II and III animals had less contraction of the patched defect and greater neomucosal surface area than Group I animals at each interval but had a lesser effect than animals receiving UG continuously. Crypt cell production rate was significantly greater in UG treated animals at 7 and 14 days but fell to control levels at 21 days. Prolonging the duration of UG infusion increased the quantity of neomucosa produced by intestinal regeneration. However, UG stimulation of mucosal cell migration and proliferation occurred transiently within 14 days after patching.
The streaming of the submandibular gland II: parenchyma and stroma advance at the same velocity
Authors: Zajicek G. , Schwartz‐Arad D. , Arber N. , Michaeli Y.
Published: May 2008

Abstract Thirty young male rats aged 7 weeks, weighing 200 g, were injected with 18.5 kBq g−1 (0.5 μCi g−1) body weight tritiated thymidine [3H]TdR (specific activity 185 GBq). The rats were then killed in groups of five, at the following times: 1 hour, and 14, 30, 60, 90, and 120 days. Autoradiograms of sections through the submandibular gland were prepared, and the location of labelled cells in tubular and acinar cross sections was recorded. The nuclear content of each cross section was defined as its ‘class’. In this numbering system, narrow tubuli, e.g. intercalated ducts are of low class, and wider tubuli, e.g. striated ducts, of high class. One hour after labelling most labelled tubular cells were found in low class cross sections, i.e. intercalated ducts and narrow granular ducts. Striated ducts were not labelled. From then onward labelled cells entered wider tubuli, e.g. striated ducts. The advancing labelled epithelium was accompanied by labelled stroma. Both cell types traversed 0.089 classes per day. In acini, labelled cells advanced in the oposite direction, starting from acinar cross sections of high class and ending in class‐1 cross sections.
Sodium‐N‐butyrate induces cytoskeletal rearrangements and formation of cornified envelopes in cultured adult human keratinocytes
Authors: Staiano‐Coico L. , Helm R. E. , McMahon C. K. , Pagan‐Charry I. , LaBruna A. , Piraino V. , Higgins P. J.
Published: May 2008

Abstract The technique developed in our laboratory allows us to culture multilayered, stratified sheets of human keratinocytes, which can be used to cover the burn wounds of patients. Organization of cells in these cultures resembles stratum germinativum and stratum spinosum but there are only a few fully keratinized cells and the stratum corneum is not developed. Since the fully differentiated sheets may offer additional advantages as epidermal transplants, attempts were made to enhance the degree of differentiation in vitro. In the present study sodium‐N‐butyrate (NaB) was used as a differentiating agent and its effect on the cell cycle and cytoarchitecture of epidermal cells was investigated.

Incubation of keratinocytes in the presence of 2.5 mM NaB induced the appearance of enucleated cornified envelopes, covering approximately 70–80% of the surface of the cultures. Their appearance correlated with a decrease in expression of keratin K13, previously shown to be inhibited during terminal differentiation of human keratinocytes. An increase in transglutaminase transferase activity was also observed. The induction of cornified layers also correlated with an increase in the amount of microfilament (MF)‐associated actin.

NaB also induced changes in the cell cycle distribution of the keratinocyte cultures. A decrease in the proportion of S and G1B phase cells was paralleled by an increase in G1A cells, maximally expressed 30–48 h following addition of the inducer. Interestingly, NaB also induced a cell arrest in G2 phase. These cell cycle perturbations preceded the onset of keratinocyte differentiation. The results indicate that the enhanced differentiation of human keratinocytes in the presence of NaB may serve as a means to produce epidermal sheets with improved properties for transplantation in a clinical setting. It also serves as an in vitro model system to study the interrelationships between biochemical events and cell cycle changes accompanying differentiation.
DNA‐synthesizing cells in oral epithelium have a range of cell cycle durations: evidence from double‐labelling studies using tritiated thymidine and bromodeoxyuridine
Authors: Hume W. J.
Published: May 2008

Abstract Mouse tongue epithelium is characterized by a circadian variation in the number of DNA‐synthesizing cells (labelling index, LI). Cells undergoing DNA synthesis were labelled with tritiated thymidine ([3H]TdR) at 0300 (peak LI) or 1200 h (low LI). The fate of these cells was assessed by injecting animals with bromodeoxyuridine (BrdU) at intervals from 12–48 h after [3H]TdR, to follow them from one cell cycle to the next. Labelling was revealed by combining [3H]TdR autoradiography with immunoperoxidase detection of BrdU in the same sections.

A single peak in the appearance of double‐labelled cells was seen at 44 h, if [3H]TdR was given at 1200 h; following [3H]TdR at 0300 h, a peak of double labelling was seen at 48 h with the possibility of smaller peaks at 24 h and 36 h.

These results show that the 24 h periodicity in LI in this tissue is associated with a predominant cell cycle duration of 44–48 h, but that a few cells cycle more quickly. Double labelling with [3H]TdR and BrdU provides a useful method for establishing cell cycle duration by labelling S‐phase cells in successive cell cycles.
Effect of sustained serum prolactin elevation on breast epithelial and myoepithelial cell proliferation
Authors: Stringer B. M. J. , Rowson J. , Greer W. , Thomas D. Wynford‐ , Williams E. D.
Published: May 2008

Abstract.

Oral administration of the dopamine antagonist perphenazine (0·01% in drinking water) to adult female Sprague‐Dawley rats led to a three‐ to fourfold increase in serum prolactin by the first time point sampled (day 2) and a sustained fourfold elevation from day 4 of treatment to the end of the experiment (day 54). In response, five‐ to sixfold (day 7) and three‐ to fourfold (day 4) peak elevations in the epithelial cell metaphase indices were seen in the breast lobular and ductular compartments respectively. Both indices fell to basal levels on day 14 but returned to a second, but diminished, peak on day 27. By day 54, the mitotic activity of the epithelium had fallen to just above basal levels in both compartments. A similar mitotic response occurred in the myoepithelial cells, clearly indicating that these must be considered an important cell kinetic component during breast stimulation. Breast epithelial cell number increased 13‐14 fold in the lobular but only two‐ to threefold in the ductular compartments in response to perphenazine administration. Again, similar responses were seen in the myoepithelial cell population. The major proliferative response therefore occurred within the lobular as opposed to the ductular compartment. A considerable discrepancy was shown between the cell number at each time point and that predicted on the assumption of constant cell death rate. We conclude that a growth desensitizing mechanism exists in the rat breast which limits breast growth in the presence of a sustained trophic hormone stimulation. Furthermore, we suggest that this limitation in breast growth is brought about by a mechanism which involves increased cell death in addition to decreased mitotic activity.
Invited review Multicell spheroids as a model for cell kinetic studies
Authors: Durand R. E.
Published: May 2008

Abstract. Cells growing in tissue culture as three‐dimensional, multicellular aggregates called ‘spheroids’ typically show a decreasing growth fraction and development of quiescent subpopulations as the spheroids enlarge. Kinetic studies in a number of spheroid systems have indicated that the primary reason for the tumour‐like growth is a progressive decrease in growth fraction, with only a modest elongation of cell cycle time in larger spheroids. In this paper, the cellular growth kinetics for spheroids of V79 Chinese hamster lung cells are reviewed, and the regrowth kinetics of cells resuming growth after recovery from quiescent regions of the spheroids are described. Further, the role of regrowth/repopulation in determining the spheroid response to anti‐tumour cytotoxics is explored, with particular emphasis on treatment with cisplatin and etoposide. By separating the effects of cytotoxicity and regrowth in the overall spheroid response to anti‐neoplastic drugs, it is suggested that ‘drug resistance’ in tumours can be a kinetic as well as a genetic problem.
Cell kinetics of a rat thyroid transplantable tumour
Authors: Christov K. , Julzary K. , Rantschev N. , Burev D.
Published: May 2008

Abstract. Changes in morphology and cell kinetics are described in a rat thyroid transplantable tumour (TTT) during the first few transplant generations. The growth of TTT in animals was possible only with an increased circulation level of the thyroid stimulating hormone (TSH). With serial transplantation subcutaneously in isologous animals, the morphology of TTT changed dramatically from that of a follicular tumour in the 3rd passage to become, by the 9th generation, a poorly differentiated tumour with a trabecular arrangement of cells. This change in tumour morphology was accompanied by an increase in the number of proliferating cells–mitotic index (MI), [3H]thymidine labelling index (LI), growth fraction (GF)–and cell loss factor (O) as well as a decrease in the cell cycle time (Tc) and potential population doubling time (TPD). TTT belongs to the class of tumours with a low proliferative activity and might be used in a variety of cell kinetic, radiobiological and chemotherapy studies.
Non‐parametric analysis of platelet lifespan
Authors: Kimmel M. , Grossi A. , Amuasi J. , Vannucchi A. M.
Published: May 2008

Abstract. A model‐independent and elementary method of analysis of platelet survival is proposed. The method is based on the finding that the mean and standard deviation of the platelet lifespan can be expressed in the terms of the slope at time 0 and the area under the empirical platelet survival curve. The method is tested using Monte‐Carlo simulations and then applied to a set of clinical data.
Tamoxifen and trifluoroperazine (Stelazine) potentiate cytostatic/cytotoxic effects of P‐30 protein, a novel protein possessing anti‐tumour activity
Authors: Mikulski S. M. , Viera A. , Ardelt W. , Menduke H. , Shogen K.
Published: May 2008

Abstract. P‐30 protein, a novel protein isolated in our laboratory from fertilized Rana pipiens eggs, has been shown to possess significant anti‐proliferative and cytotoxic activity against a variety of human tumour cell lines. This protein also shows a potent anti‐tumour activity in vivo in animal tumour models and is currently undergoing Phase I human clinical trials in cancer patient volunteers. The present study describes the in vitro effects of the concerted action of this protein and two other agents which affect the cell proliferative cycle. A significant potentiation of the P‐30 protein‐induced cell growth inhibition by tamoxifen as well as trifluoroperazine (Stelazine) in both the human A‐549 lung carcinoma and the ASPC‐1 pancreatic adenocarcinoma systems at wide ranges of drug concentrations was observed. The effect was apparently due to the synergistic action of P‐30 protein and the agents tested. This data may provide clues that can be useful in explaining the mechanism of its anti‐tumour activity. The results are also helpful for the designing in vivo animal and, perhaps eventually, human studies, whereby the combination therapies utilizing P‐30 protein with agents of relatively low toxicity such as tamoxifen and/or Stelazine could offer a promising treatment(s) for these notoriously refractory types of human cancer.
Is there an accumulation of cells during G2 in some acute lymphoblastic leukaemias?
Authors: Ffrench M. , Magaud J. P. , Arzounian M. , Souchier C. , Charrin C. , Bryon P. A.
Published: May 2008

Abstract. In some cases of acute lymphoblastic leukaemia (ALL) the percentage of cells in G2+ M is higher than anticipated when compared with the percentage in S phase. This increase in G2+ M, as detected by flow cytometry measurement of DNA content, may be due to an accumulation of cells, either in G 2 or during the end of S phase; it may also be related to the existence of small tetraploid clones generally ignored by cytogeneticists. In order to identify possible subpopulations of cells with a DNA index ≥ 2‐0, we have compared the results of a cytogenetic analysis to the G2+ M values. We have also studied the distribution of S phase cells in 24 cases of ALL by incorporating 5‐bromodeoxyuridine, labelling the cells by indirect immunofluorescence, and analysing them by flow cytometry after propidium iodide staining. The distribution of cells during S phase was quantified: no accumulation of cells was ever observed at the end of S phase. The question of the existence of small tetraploid clones, G2 arrested cells or cells with a G2 elongation remains open. However, we feel that it is more probable that, in this pathology, an elongation of the duration of G2 occurs.
Tetraploid cycle in ageing solid tumours
Authors: Liautaud‐Roger F. , Dufer J. , Coninx P.
Published: May 2008

Abstract. When the mouse mammary adenocarcinoma 755 (Ca‐755) reaches the plateau phase of growth, non‐cycling cells with a G2‐DNA content can be observed. They may belong to the diploid cell cycle but they could also be blocked in G0 or G1 of a tetraploid cycle. This hypothesis was tested in three ways: (1) non‐cycling G2 nuclei were stained with a combination of Feulgen and naphthol yellow which revealed two populations, one with a low protein content and the other with a high protein content– the latter may represent nuclei ready to begin a new phase of DNA synthesis; (2) Feulgen staining and autoradiography were performed after tritiated thymidine had been administered to mice continuously: this showed that there were cells synthesizing DNA with a DNA index above 2; and (3) cells having 80 chromosomes, corresponding to the tetraploid cycle, were found almost exclusively in the plateau phase tumours.

On the other hand, the use of texture and DNA parameters of the Feulgen stained nuclei showed that they were concentrated in a diploid cycle for tumours in the exponential phase of growth and were divided between a diploid and tetraploid cycle for ‘plateau’ cells. Neither the cause for, nor the role played by, polyploid cells is known.
Double labelling of cells with tritiated thymidine and bromodeoxyuridine reveals a circadian rhythm‐dependent variation in duration of DNA synthesis and S phase flux rates in rodent oral epithelium
Authors: Hume W. J. , Thompson J.
Published: May 2008

Abstract. We describe a double labelling method for estimating the duration of DNA synthesis (Ts) and the flux of cells into and from the S phase of the cell cycle, based on labelling with tritiated thymidine ([3H]TdR) followed by bromodeoxyuridine (BrdU) and combining immunohistological detection of BrdU with conventional autoradiography. In practice, the change in size of a window of double labelled cells occurs as the time interval between the two labels increases. In mouse tongue epithelium there is a marked circadian variation in the number of cells in DNA synthesis. From 0900 to 1500 h this labelling index (LI) falls, but from 2100 to 0300 h it increases. Our results show that the circadian decrease in LI is associated with a short Ts (5·8 ± 0·3 h), a high S phase efflux and an initially low influx of cells from G: into S. Conversely, the rising circadian LI is associated with a longer Ts (9.4 ± 0.1 h), an initially low efflux and a moderate to high influx. Two time‐points exist on the circadian LI curve when influx and efflux rates change abruptly. At 0100 h the efflux rate rises from low (5 cells %/h) to high (15–16 cells %/h) and simultaneously the influx rate changes from high to low. Similarly at 1300–1400 h, efflux rate falls from high (19–20 cells %/h) to low (4–8 cells %/h) values and influx rates change from low to high. This double labelling method has revealed that the duration of DNA synthesis varies across the circadian cycle, as do influx and efflux values which generally fall within a discrete range of high or low values. The timing of the changes in flux suggests the presence of two ‘control’ points on the circadian LI cycle that were previously unrecognized.
Proliferative activity of primary breast cancer and of synchronous lymph node metastases evaluated by [3H]‐thymidine labelling index
Authors: Daidone M. G. , Silvestrini R. , Valentinis B. , Persici P. , Mezzanotte G. , Squicciarini P. , Orefice S. , Salvadori B.
Published: May 2008

Abstract. The [3H]‐thymidine labelling index ([3H]TdR LI) has been used to evaluate and comparatively analyse the proliferative activity of different tumour lesions from the same patient. The analysis was performed on the primary tumour and its synchronous lymph node metastasis from 210 patients operated on for breast cancer. A direct relation was observed between the proliferative activity of the two different lesions (Spearman correlation coefficient = 0–46, P<00001), but there was considerable scatter amongst the data. The [3H]TdR LI of primary and of metastatic lesions belonged to the same proliferation classes in only 47% of the cases. Higher or lower [3H]TdR LI values, categorized on the basis of the tertiles of the frequency distribution, occurred in the node metastasis than in the primary tumour in an almost similar percentage of the remaining cases. Menopause, receptor status and pathological features did not affect interlesion kinetic patterns. The prognostic role of the proliferative activity of the two different lesions was investigated on 107 patients with stage II tumours homogeneously treated with surgery and systemic adjuvant therapy. Relapse‐free survival at 3 years was significantly affected by the proliferative activity of the primary tumour but not by that of the lymph node metastasis.
Mutagenic effects of brief exposure to bromodeoxyuridine on mouse FM3A cells
Authors: Matsuoka K. , Nomura K. , Hoshino T.
Published: May 2008

Abstract. The time‐ and dose‐dependency of the mutagenic effects of bromodeoxyuridine (BrdU), a thymidine analogue used for cell kinetics studies in vivo and in vitro, were investigated in FM3A cells. Cells incubated with 50–1000 fin BrdU for 72 h showed some inhibition of growth. Cells cultured in BrdU‐free medium for 3 d after a 30 min or 2 h exposure to BrdU showed no growth inhibition, while those previously exposed for 24 h to BrdU showed retarded growth. After a 30 min exposure, 60% of cells were labelled with BrdU; after 2 h 70%; and after 24 h almost 100%. After incubation in BrdU‐free medium for 3 d (the time required for this cell line to express mutation), cells previously treated for 30 min or 2 h showed reduced BrdU positivity, whereas almost 100% of those treated for 24 h remained BrdU positive. The mutation rate, determined by the number of colonies resistant to ouabain (2 mM) and 6‐thioguanine (10 μ) 3 d after exposure to BrdU, was not affected by a 30 min treatment with up to 1000 μ BrdU. Cells treated for 1 or 2 h showed increased resistance to ouabain after exposure to BrdU at concentrations above 100 μM; cells treated for 12 or 24 h showed an increased mutation rate at BrdU concentrations above 50 μM… The number of colonies resistant to 6‐thioguanine did not increase in cells treated with BrdU at concentrations up to 1000 μM for 1, 12 or 24 h. We cannot conclude with certainty that brief exposure to BrdU does not modulate DNA to the point of mutation. This study may serve as a guideline for limiting the dose and time of exposure to BrdU for cell kinetics studies in vivo and in vitro.
Different nucleolar antigen expression in resting and proliferating human lymphocytes as studied by fluorescence microscopy and flow cytometry
Authors: Dubben H.‐H.
Published: May 2008

Abstract. The aim of this study was to investigate the use of a nucleolar antigen to discriminate between proliferating and resting cells. Antinucleolar antibodies (Si87) were obtained from a scleroderma patient. The specificity of immunostaining was verified and morphological changes in nucleoli were monitored using a fluorescence microscope. Fluorescence of propidium iodide‐stained DNA and nucleolar immunofluorescence were measured by flow cytometry.

Following phytohaemagglutinin stimulation the number of nucleoli of normal human peripheral blood lymphocytes increased about 3‐fold, accompanied by enlargement of nucleolar size. Simultaneously a mean increase in total immunofluorescence per cell by a factor of three was detected. The method developed and applied here allows a discrimination between resting and proliferating human lumphocytes on the basis of their nucleolar antigen content.
Protein phosphorylation in developing and regenerating rat kidney
Authors: Hoang T. , Bergeron M.
Published: May 2008

Abstract. Renal cytosolic extracts from rats of different ages and mononephrectomized rats were incubated with γ‐[32P]ATP and analysed by high resolution two‐dimensional electrophoresis and autoradiography. Extracts from new‐born and young rats showed a great number of phosphorylated proteins migrating between the origin and Mr 52,000. Among these proteins, the group co‐migrating with phosphorylase b (Mr 97,000) was particularly evident in new‐born and days‐old rats. In extracts from mature rats, other proteins of lower molecular weight, particularly those migrating between Mr 60,000 and 44,000, became intensely phosphorylated. The number and intensity of phosphorylated proteins from extracts of normal and nephrectomized rats, however, did not vary. Activity of cAMP‐dependent protein kinase and [3H]cAMP binding was also modified during neonatal development but not in compensatory renal growth. Since cAMP‐PK and protein phosphorylation are known to be regulated in response to hormonal stimulations, these results may provide good indications for the understanding of hormonal involvement in kidney growth.
Age‐related changes in the cell kinetics of rat foot epidermis
Authors: Morris G. M. , van den Aardweg G. J. M. J. , Hamlet R. , Whitehouse E. , Hopewell J. W. , Franke H. , Loeffler M.
Published: May 2008

Abstract. The durations of the cell cycle and its component phases have been determined for the basal layer of the epidermis of the skin from the upper surface of the hind foot of the rat using single pulse [3H]‐thymidine labelling and the percent labelled mitosis (PLM) technique. Rats of three age groups were used, namely 7, 14 and 52 weeks. The duration of DNA synthesis (Ts) and the G2 plus M phase (Tg2± m) were comparable in 7‐week and 52‐week‐old rats (P > 0–1). The major difference between 7‐week and 52‐week‐old rats was in the duration of the G1 phase (Tg1). In 7‐week‐old rats Tg1 was 15.0 ± 0.8 h and in 52‐week‐old rats Tg1 was 31.2 ± 3.5 h. A consequence of this variation was that the overall duration of the cell cycle was longer in 52‐week‐old rats (53.9 ± 5.3 h) than in 7‐week‐old rats (30.1 ± 1.3 h).

Difficulties were found in fitting a simple curve to the PLM data for 14‐week‐old rats. This suggests that the proliferative cell population of the epidermis of rats of this age group may be heterogeneous. A satisfactory fit to the data was obtained using a computer model which assumed that the proliferative population of the epidermis of 14‐week‐old rats was a mixture of cells with cell cycle parameters the same as those of the 7‐week and the 52‐week‐old rats. These two sub‐populations of relatively slowly and rapidly proliferating cells were present in the ratio of 2:1.
Cell proliferation kinetics of six xenografted human cervix carcinomas: comparison of autoradiography and bromodeoxyuridine labelling methods
Authors: van Oostrum I. E. A. , Rozemuller E. , Knol R. F. G. , Erkens‐Schulze S. , Rutgers D. H.
Published: May 2008

Abstract. Cell kinetic and histologic parameters of six xenografted tumours with volume doubling times ranging from 6 to 43 d were investigated in order to obtain kinetic information on a panel of tumours to be used in radiobiological studies. The six tumours covered a range of histologies and their DNA indices varied from 2–7 to 1–4. The length of the cell cycle (Tc), potential doubling time (Tpot) and labelling index (LI) were determined by continuous labelling with [3H]TdR and autoradiography in three tumours. Tc varied from 30 to 40 h. Determinations of the length of the S phase (Ts) were found to be less reliable by this method.

Data on Ts and LI were also determined in all six tumours using bromodeoxyuridine (BrdU) labelling and the single sample method; values of Tpot were slightly longer than those obtained via the autoradiographic method. In addition, multiple samples were taken after BrdU labelling. Tc was determined by fitting the data obtained from mid‐S, mid‐G2 and mid‐G1 windows to curves described by a damped oscillator. Data obtained via the mid‐S window were found to be most reliable. Generally, cell cycle times obtained by the BrdU method were longer than those observed with the autoradiographic method. Differences between the two methods could be explained by inaccuracies in the determination of Ts, LI and Tc and differences in the experimental approach.

We consider the BrdU labelling method to be a suitable alternative for the time‐consuming autoradiography, if data on Ts or Tpot are sufficient. Due to difficulties in the reproducibility of the immunofluorescence staining and asynchronization of cells approximately 10 h after labelling, the method of windows analysis was affected by similar problems to those observed in interpretation of percentage labelled mitosis (PLM) curves. However, the method may serve as an alternative to determine cell cycle times in vitro and, if improved technically, in vivo. Careful comparison of the data obtained from mid‐S, mid‐G1 and mid‐G2 windows may increase the reliability of the determination of cell kinetic parameters.
Lack of correlation between thymidine kinase activity and changes of DNA synthesis with tumour age: an in vivo study in Ehrlich ascites tumour
Authors: Skog S. , He Q. , Tribukait B.
Published: May 2008

Abstract. Thymidine kinase (TK) and its isoenzymes were studied in relation to age of Ehrlich ascites tumour cells growing in vivo. Various steps of the pathway of thymidine through deoxynucleotide metabolism were studied: [3H]‐thymidine cellular uptake and incorporation into DNA; the cellular nucleotide pools; and the concentration of thymidine in ascites. In addition, the proportion of cells in the various parts of the cell cycle and the bromodeoxyuridine labelling index were determined.

Four isoenzymes at pi 41, 5‐3, 6–9 and 8‐3 were identified using isoelectric focusing. The TK activity declined with age of the tumour by about 90%, mostly due to a decrease of the isoenzyme at pi 8‐3. However, this decline was neither related to the changes in DNA synthesis rate of the cells with tumour age, nor to the proportion of cells in S‐phase or the bromodeoxyuridine (BrdU) labelling index. In contrast, the contribution of DNA synthesis via the thymidine salvage pathway relative to the total DNA synthesis increased from less than 1% at exponential growth to about 15% at plateau phase of growth. Blocking of DNA synthesis by aphidicolin did not change the TK activity. We therefore conclude that changes in TK activity and changes in cell growth are epiphenomena rather than causally related to each other.

All nucleotide pools decreased with tumour age. The inhibition of TK by an increase in the deoxythymidine triphosphate pool could therefore be excluded. With a decrease of the TK activity during tumour growth, increasing amounts of TdR were excreted by the cells and accumulated in the ascites fluid. To explain our results on TK activity we propose a substrate cycle in which thymidine monophosphate supplied by de novo synthesis is dephosphorylated and is then either phosphorylated by TK to thymidine monophosphate or excreted by the cell.
Observations regarding DNA replication sites in human cells in vivo following infusions of iododeoxyuridine and bromodeoxyuridine
Authors: Raza A. , Miller M. A. , Mazewski C. , Sheikh Y. , Lampkin B. , Sawaya R. , Crone K. , Berger T. , Reising J. , Gray J. , Khan S. , Preisler H.
Published: May 2008

Abstract. In studies using bromodeoxyuridine (BrdUrd) and/or iododeoxyuridine (IdUrd) to label S phase cells in cancer patients, several unique observations were made regarding DNA replication sites and the organization of newly synthesized DNA in post‐mitotic cells. While the majority of tumour specimens removed at the end of infusions demonstrated concentration of replication sites around the nuclear membrane, biopsies obtained in leukaemic patients 1 week later demonstrated several distinct patterns of labelling. For example, one, two or all lobes of granulocytes were labelled. Scavenger macrophages bearing labelled leukaemic cells in their cytoplasm were also seen. Sequential IdUrd/BrdUrd labelling of solid tumours showed various patterns of nuclear/nucleolar/membrane labelling, allowing more precise localization of early versus late replication sites.
Inhibited morphological terminal differentiation and enhanced proliferation of cultured mouse epidermal cells at different concentrations of dimethyl sulphoxide
Authors: Miller D. R. , Allison D. P. , Rorvik M. C. , Slagat T. J.
Published: May 2008

Abstract. Dimethyl sulphoxide (DMSO), at concentrations of 1–2%, induces terminal differentiation in several different cell types in vitro and enhances the growth of newborn mouse epidermal cells in primary culture under conditions that also permit terminal differentiation. We have found that DMSO concentrations approaching 4% reversibly inhibited (with little overt toxicity) terminal differentiation of normal epidermal cells from newborn SENCAR mice. Cells cultured in medium containing 4% DMSO and calcium in excess of 1 mm did not stratify extensively or slough large amounts of keratinized debris into the medium as occurred in control cultures, nor did they form large numbers of squamous cells or keratin bundles, as revealed by light and electron microscopy. The number of detergent‐insoluble cornified envelopes was similarly reduced. Long‐term growth of epidermal colonies in secondary culture was optimum in 1% DMSO, this concentration also permitting normal terminal differentiation of these cells. Since DMSO had these effects on epidermal cells in vitro, it may also affect epidermal cell proliferation and terminal differentiation in vivo, an important consideration should DMSO ever be approved for topical use in the US.
Dose‐dependent induction of apoptosis in human tumour cell lines by widely diverging stimuli
Authors: Lennon S. V. , Martin S. J. , Cotter T. G.
Published: May 2008

Abstract. Cell death may occur by either of two mechanisms: apoptosis or necrosis. Necrosis, the first type of cell death to be recognized, is an uncontrolled degenerative phenomenon invariably caused by noxious stimuli and is the result of irreversible failure of membrane function. Apoptosis, on the other hand, is a death process which involves a series of well‐organized events which require active cell participation, and is primarily caused by physiological stimuli. In the present study we show that cell death induced by a range of varied agents may take the form of either apoptosis or necrosis. Apoptotic cell death was found to occur at low levels of these agents, while at higher levels necrosis occurred. Hence, cells which are not killed directly, but merely injured by these agents, have the capacity to activate an internally programmed suicide death mechanism, whereas cells receiving greater injuries apparently do not. In addition, the presence of extracellular calcium was found to be necessary for the induction of apoptosis with all agents tested.
Distribution of Adriamycin in mice bearing mammary adenocarcinoma 16/C
Authors: Simpson‐Herren L. , Noker P. E.
Published: May 2008

Abstract. The response of solid mammary adenocarcinoma 16/C to treatment with Adriamycin is highly variable and ranges from growth under treatment to complete regression. Tumour and host factors were evaluated to determine the influence of each on the response. We determined that the concentration of Adriamycin in plasma and tumour was a function of tumour size and treatment history in mice bearing mammary adenocarcinoma 16/C. The plasma concentrations following a single dose of Adriamycin (10 mg/kg) increased in proportion to tumour mass without a concurrent increase in tumour concentration. When mice bearing large tumours (>1·0 g) were treated with a multidose protocol, the plasma concentrations were higher and the tumour concentrations lower following the initial dose than following subsequent doses; in tumour‐free mice, prior treatment with Adriamycin did not affect the plasma level achieved after a second dose. The magnitude of the decrease in plasma and increase in tumour concentrations was a function of the initial tumour size and the treatment schedule. The increase in tumour levels represented the sum of residual Adriamycin and drug bound as a result of the dose immediately prior to analysis. At the time of the initial treatment, the Adriamycin was distributed within each tumour in proportion to vascular perfusion. The percent of the tumour mass that was well‐perfused appeared to increase with repeated treatments. The results indicate that the plasma concentration of Adriamycin did not pecessarily reflect the tumour exposure in the mammary adenocarcinoma 16/C model. In hosts bearing mammary adenocarcinoma 16/C—or, possibly, other tumours that produce similar effects on the host—a low initial dose of Adriamycin might modify the distribution, possibly reduce the toxicity and allow escalation of subsequent doses with increased exposure of the tumour.
Measuring cell proliferation by relative movement. I. Introduction and in vitro studies
Authors: White R. A. , Terry N. H. A. , Baggerly K. A. , Meistrich M. L.
Published: May 2008

Abstract. This paper presents two new ways of analysing data which may be obtained from pulse labelling a population of cells with bromodeoxyuridine and analysing that population as a function of time with bivariate flow cytometry. The progression of cells is measured by the change in position in the cell cycle, as shown by a change in the mean DNA content of the labelled and unlabelled cells. The particular measures of the mean DNA content used are extensions of the relative movement of the labelled undivided cells, RMlu(t), which was introduced by Begg and co‐workers to measure the DNA synthesis time, Ts. In general, the relative movement is defined as the mean DNA fluorescence of a population of cells less the DNA fluorescence of the cells in G1 and divided by the difference in DNA fluorescence of the cells in G2+ M and G1. In this paper we examine the relative movements of all the labelled cells and all of the unlabelled cells, denoted RML(t) and RMU(t) respectively. It is found that RML(t) and RMU(t) exhibit clear cyclic behaviour and distinguishable characteristics which depend directly on the transit times (T) of the cell cycle phases, i.e. TG1, Ts and TG2+m. Furthermore, the peak heights of the RMU(t) curve are shown to depend strongly on the growth fraction of the population under consideration. A theoretical treatment of the curves so obtained is presented, and is shown to yield values in close agreement with those from other methods for measuring these transit times and a lower limit to values for the growth fraction of Chinese hamster ovary cells grown in vitro.
Differentiation of HL‐60 myeloid leukaemia cells is associated with a transient block in the G2 phase of the cell cycle
Authors: Laskin D. L. , Sirak A. A. , Laskin J. D.
Published: May 2008

Abstract. The human promyelocytic leukaemia cell line HL‐60 can be induced to differentiate towards mature granulocytes by treatment with dibutyryl cyclic adenosine‐3′,5′‐monophosphate (dbcAMP). Differentiation begins within 16–24 h of treatment and is associated with a time‐ and dose‐dependent accumulation of cells in the G0/G1 phase of the cell cycle with a concomitant decrease in the number of cells in the S and G2+ M phases. Using acridine orange staining, we found that the RNA content of the cells also decreased following differentiation. Stathmokinetic analysis of HL‐60 cell populations following dbcAMP treatment showed no effect on the total number of cells in the G0/G1 or S phases, or the rate of progression of cells through these cell cycle compartments. In contrast, dbcAMP was found to induce a transient arrest of the cells in the G2 phase. We also found that differentiation induced by dbcAMP did not require progression of the cells through the cell cycle. Cells arrested in either G1/S by hydroxyurea or G2+ M by colcemid eventually expressed markers of mature granulocytes. These results demonstrate that dbcAMP modulates cell cycle progression. However, these cell cycle changes alone are insufficient to induce granulocytic differentiation of HL‐60 cells.
Cell cycle status of stromal cells in long‐term haematopoietic cultures
Authors: Choudhury C. , Sparks R.
Published: May 2008

Abstract. This study was performed to further define the mechanism by which the stromal micro‐environment regulates haematopoiesis. In long‐term marrow cultures the interactions between stromal cells and haematopoietic cells can be investigated at the cellular level. Long‐term marrow cultures from hamsters do not require repopu‐lation or addition of hydrocortisone and are suitable for investigation of cell kinetics. The cellular kinetics of haematopoietic and stromal cells, as studied by tritiated thymidine ([3H]dT) incorporation, revealed that DNA synthesis occurred in both the non‐adherent and the adherent cells. In established cultures the adherent stromal cells were predominantly in a quiescent non‐cycling state: <2% adherent cells incorporated [3H]dT within 5 h. Removal of the supernatant cells did not affect the labelling index of adherent cells, since the labelling indices at the 50–75 h time point were 14·3% and 12·5% in the presence and absence of supernatant cells respectively. An apparent stimulus for stromal cells to incorporate [3H]dT was attachment or adhesion. Following replating of supernatant cells of long‐term marrow cultures, 23·3% of the reformed adherent layer cells were labelled compared with 12–14% in cultures with previously formed unmobilized adherent cells (P < 0·01). The data indicate that adherent cells are not required to synthesize DNA for maintenance of haematopoiesis in established long‐term marrow cultures, and that recruitment into the cell cycle has an independent mechanism that is not influenced by feed‐back from the supernatant cells.